LOCUS       sequence-412886-        6922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses modified cytolysin S (mCylLS) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6922)
  AUTHORS   Luo Y, Xu S, Frerk AM, van der Donk WA
  TITLE     Facile Method for Determining Lanthipeptide Stereochemistry.
  JOURNAL   Anal Chem. 2024 Jan 30;96(4):1767-1773. doi: 
            10.1021/acs.analchem.3c04958. Epub 2024 Jan 17.
  PUBMED    38232355
REFERENCE   2  (bases 1 to 6922)
  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"; doi: 
            "10.1021/acs.analchem.3c04958"; journalName: "Anal Chem"; date: 
            "2024-01-30- 30"; volume: "96"; issue: "4"; pages: "1767-1773"
COMMENT     Sequence Label: pRSFDuet1_CylLS_CylM
FEATURES             Location/Qualifiers
     source          1..6922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(187..1269)
                     /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     1231..1250
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1270..1347)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1393..1411
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1412..1436
                     /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             1492..1509
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1782..1800
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1801..1825
                     /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             4868..4912
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4950..4968)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4964..5011
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5241..6056)
                     /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(5241..6056)
                     /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     5964..5983
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6057..6148)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6164..6913)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(6174..6191)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6414..6433)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
       61 gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
      121 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
      181 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
      241 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
      301 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
      361 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
      421 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
      481 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
      541 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
      601 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
      661 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
      721 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
      781 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
      841 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
      901 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
      961 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     1021 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     1081 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     1141 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     1201 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     1261 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     1321 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     1381 gcattaggaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctg
     1441 tagaaataat tttgtttaac tttaataagg agatatacca tgggcagcag ccatcaccat
     1501 catcaccaca gccaggatcc gaattcgctg aataaagaaa atcaggaaaa ttattatagc
     1561 aataaactgg aactggtggg tccgagcttt gaagaactga gcctggaaga aatggaagca
     1621 attcagggta gcggtgatgt tcaggcagaa accacaccgg catgttttac cattggtctg
     1681 ggtgttggtg cactgtttag cgcaaaattt tgctaagcgg ccgcataatg cttaagtcga
     1741 acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg
     1801 ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga
     1861 tatacatatg gaagataatc tgattaatgt gctgagcatt aatgaacgtt gctttctgct
     1921 gaaacagagc ggcaaagaaa aatatgatat taaaaatctg caggcctgga aagaacgtaa
     1981 aagcgttctg aaacaggatg atctggatta tctgattaaa tataaatatg aaagcctgga
     2041 taattttggc ctgggtatta ccccgattga aaattttccg gataaagaag tggccattca
     2101 gtatattaaa gatcagagtt ggtatatttt ttttgaaagc attctggata gctataatga
     2161 tagcgaagaa aaactgctgg aagttgatgc aagctatccg tttcgttatt ttctgcagta
     2221 tgcacgtctg tttctgctgg atctgaatag cgaactgaat atttgcacca aagaatttat
     2281 tattaatctg ctggaaaccc tgacccagga actgattcat ctgaccagca aaaccctggt
     2341 tctggatctg cataccttta aaaaaaatga accgctgaaa ggcaatgata gcagcaaacg
     2401 ctttatttat tatctgaaaa aacgctttaa tagcaaaaaa gatattattg ccttttatac
     2461 ctgctatccg gaactgatgc gtattaccgt tgttcgtatg cgctatttcc tggataacac
     2521 caaacaaatg ctgattcgtg ttaccgaaga tctgccgagc attcagaatt gctttaatat
     2581 tcagagcagt gaactgaata gcattagcga aagccagggt gatagccata gccgtggtaa
     2641 aaccgttagc accctgacct ttagtgatgg taaaaaaatt gtgtataaac cgaaaattaa
     2701 tagcgaaaat aaactgcgcg atttttttga atttctgaat aaagaactgg aagccgatat
     2761 ttatattgtg aaaaaagtga cccgcaatac ctatttttat gaagaatata tcgataacat
     2821 tgaaatcaat aacatcgaag aagtgaaaaa atattacgaa cgctatggca aactgattgg
     2881 cattgccttt ctgtttaatg ttaccgatct gcattatgaa aacatcattg cccatggcga
     2941 atatccggtg attattgata atgaaacctt ttttcagcag aatattccga ttgaatttgg
     3001 taatagcgca accgttgatg ccaaatacaa atatctggat agcattatgg tgaccggtct
     3061 ggttccgtat ctggcaatga aagataaaag cgatagcaaa gatgaaggcg ttaatctgag
     3121 cgcactgaat tttaaagaac agagcgtgcc gtttaaaatt ctgaaaatta aaaatacctt
     3181 taccgatgaa atgcgctttg aatatcagac ccatattatg gataccgcaa aaaatacccc
     3241 gattatgaat aatgaaaaaa tcagctttat cagctatgaa aaatatattg tgaccggcat
     3301 gaaaagcatt ctgatgaaag ccaaagatag caaaaaaaaa attctggcct atattaataa
     3361 taatctgcag aatctgattg tgcgcaatgt tattcgtccg acccagcgtt atgcagatat
     3421 gctggaattt agctatcatc cgaattgctt tagcaatgcc attgaacgtg aaaaagtgct
     3481 gcataatatg tgggcctatc cgtataaaaa taaaaaagtg gtgcattatg aattttcaga
     3541 tctgattgat ggcgatattc cgatttttta taataatatt agcaaaacca gcctgattgc
     3601 cagtgatggt tgtctggttg aagattttta tcaggaaagt gccctgaatc gctgcctgaa
     3661 taaaattaat gatctgtgtg atgaagatat tagcattcag accgtgtggc tggaaattgc
     3721 actgaatatt tataacccgt acaaatatat caatgatctg aaaaaccaga atagcaacaa
     3781 atatatttat accggtctgg aactgaatgg caaaattatt caggcctgcc agaaaattga
     3841 aaaaaaaatc tttaaacgtg ccatctttaa caaaaaaacc aataccgtga attggattga
     3901 tattaaactg gatcaggatt ggaatgtggg cattctgaat aataatatgt atgatggcct
     3961 gcctggtatt tttatttttt atgtggccct gaaatatatt accaaaaacc ataaatatga
     4021 ttatgtgatc gaatgcatta aaaatagcat ttataccatt ccgagcgaag atattctgag
     4081 cgcatttttt ggtaaaggca gcctgattta tccgctgctg gttgattatc gcctgaataa
     4141 tgatattaat agcctgaatg tggccgtgga aattgccgat atgctgattg aaaaaaaacc
     4201 gattaataat ggcgaactga aaaatgattg gattcatggc cataatagca ttattaaagt
     4261 gctgctgctg ctgagcgaaa ttaccgaaga tgaaaaatat cgcaaattta gcctggaaat
     4321 ttttgaaaaa ctgagcgaag aaccgtattt taattttcgt ggttttggcc atggcattta
     4381 tagctatgtt catctgctga gcaaatttaa tcgcattgat aaagccaata gcctgctgca
     4441 taaaattaaa gaaagctatt ttgaagaaga accgaaaaat aattcctggt gtaaaggcac
     4501 cgttggtgaa ctgctggcaa ccattgaact gtatgatgat aatattagta acatcgatat
     4561 taacaaaacc attgcctata aaaataaaga ttgcctgtgc catggcaatg caggcaccct
     4621 ggaaggtctg attcagctgg caaaaaaaga tccggaaacc tatcagtata aaaaaaataa
     4681 actgatcagc tatatgctga atgattttga aaaaaataat accctgaaag tggcaggcag
     4741 cgaatatctg gaaagcctgg gtttttttgt tggtattagc ggtgttggtt atgaactgct
     4801 gcgtaatctg gatagcgaaa ttccgaatgc actgctgttt gaactgtaag gtaccctcga
     4861 gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact cgtctactag
     4921 cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     4981 ggcctctaaa cgggtcttga ggggtttttt gctgaaacct caggcatttg agaagcacac
     5041 ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat
     5101 ttaacgaccc tgccctgaac cgacgacaag ctgacgaccg ggtctccgca agtggcactt
     5161 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5221 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     5281 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     5341 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     5401 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     5461 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     5521 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     5581 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct gttaaaagga
     5641 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     5701 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     5761 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     5821 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     5881 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     5941 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     6001 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcatactc
     6061 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6121 tttgaatgta tttagaaaaa taaacaaata ggcatgcagc gctcttccgc ttcctcgctc
     6181 actgactcgc tacgctcggt cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg
     6241 gtaatacggt tatccacaga atcaggggat aaagccggaa agaacatgtg agcaaaaagc
     6301 aaagcaccgg aagaagccaa cgccgcaggc gtttttccat aggctccgcc cccctgacga
     6361 gcatcacaaa aatcgacgct caagccagag gtggcgaaac ccgacaggac tataaagata
     6421 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6481 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6541 ttggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6601 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6661 acacgactta tcgccactgg cagcagccat tggtaactga tttagaggac tttgtcttga
     6721 agttatgcac ctgttaaggc taaactgaaa gaacagattt tggtgagtgc ggtcctccaa
     6781 cccacttacc ttggttcaaa gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg
     6841 tagcggtggt ttttctttat ttatgagatg atgaatcaat cggtctatca agtcaacgaa
     6901 cagctattcc gttactctag at
//
