LOCUS       sequence-412889-        6937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses modified cytolysin L (mCylLL) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6937)
  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 6937)
  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_CylLL_CylM
FEATURES             Location/Qualifiers
     source          1..6937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(220..1302)
                     /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     1264..1283
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1303..1380)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1426..1444
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1445..1469
                     /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             1525..1542
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1830..1848
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1849..1873
                     /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             4916..4960
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4998..5016)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5012..5059
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5289..6104)
                     /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(5289..6104)
                     /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     6012..6031
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6105..6196)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(6212..6937,1..24))
                     /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(6222..6239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6462..6481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aagtcaacga acagctattc cgttactcta gatttcagtg caatttatct cttcaaatgt
       61 agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
      121 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
      181 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
      241 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
      301 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
      361 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      421 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
      481 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
      541 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
      601 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      661 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      721 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      781 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
      841 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      901 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      961 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     1021 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     1081 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     1141 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     1201 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     1261 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     1321 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     1381 cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata cgactcacta
     1441 taggggaatt gtgagcggat aacaattccc ctgtagaaat aattttgttt aactttaata
     1501 aggagatata ccatgggcag cagccatcac catcatcacc acagccagga tccgaattcg
     1561 gaaaatctga gcgttgttcc gagctttgaa gaactgagcg ttgaagaaat ggaagcaatt
     1621 cagggtagcg gtgatgttca ggcagaaacc acaccggttt gtgcagttgc agcaaccgca
     1681 gcagcaagca gcgcagcatg tggttgggtt ggtggtggta tttttaccgg tgttaccgtt
     1741 gttgttagcc tgaaacattg ctaagcggcc gcataatgct taagtcgaac agaaagtaat
     1801 cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg aattgtgagc
     1861 ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata tacatatgga
     1921 agataatctg attaatgtgc tgagcattaa tgaacgttgc tttctgctga aacagagcgg
     1981 caaagaaaaa tatgatatta aaaatctgca ggcctggaaa gaacgtaaaa gcgttctgaa
     2041 acaggatgat ctggattatc tgattaaata taaatatgaa agcctggata attttggcct
     2101 gggtattacc ccgattgaaa attttccgga taaagaagtg gccattcagt atattaaaga
     2161 tcagagttgg tatatttttt ttgaaagcat tctggatagc tataatgata gcgaagaaaa
     2221 actgctggaa gttgatgcaa gctatccgtt tcgttatttt ctgcagtatg cacgtctgtt
     2281 tctgctggat ctgaatagcg aactgaatat ttgcaccaaa gaatttatta ttaatctgct
     2341 ggaaaccctg acccaggaac tgattcatct gaccagcaaa accctggttc tggatctgca
     2401 tacctttaaa aaaaatgaac cgctgaaagg caatgatagc agcaaacgct ttatttatta
     2461 tctgaaaaaa cgctttaata gcaaaaaaga tattattgcc ttttatacct gctatccgga
     2521 actgatgcgt attaccgttg ttcgtatgcg ctatttcctg gataacacca aacaaatgct
     2581 gattcgtgtt accgaagatc tgccgagcat tcagaattgc tttaatattc agagcagtga
     2641 actgaatagc attagcgaaa gccagggtga tagccatagc cgtggtaaaa ccgttagcac
     2701 cctgaccttt agtgatggta aaaaaattgt gtataaaccg aaaattaata gcgaaaataa
     2761 actgcgcgat ttttttgaat ttctgaataa agaactggaa gccgatattt atattgtgaa
     2821 aaaagtgacc cgcaatacct atttttatga agaatatatc gataacattg aaatcaataa
     2881 catcgaagaa gtgaaaaaat attacgaacg ctatggcaaa ctgattggca ttgcctttct
     2941 gtttaatgtt accgatctgc attatgaaaa catcattgcc catggcgaat atccggtgat
     3001 tattgataat gaaacctttt ttcagcagaa tattccgatt gaatttggta atagcgcaac
     3061 cgttgatgcc aaatacaaat atctggatag cattatggtg accggtctgg ttccgtatct
     3121 ggcaatgaaa gataaaagcg atagcaaaga tgaaggcgtt aatctgagcg cactgaattt
     3181 taaagaacag agcgtgccgt ttaaaattct gaaaattaaa aataccttta ccgatgaaat
     3241 gcgctttgaa tatcagaccc atattatgga taccgcaaaa aataccccga ttatgaataa
     3301 tgaaaaaatc agctttatca gctatgaaaa atatattgtg accggcatga aaagcattct
     3361 gatgaaagcc aaagatagca aaaaaaaaat tctggcctat attaataata atctgcagaa
     3421 tctgattgtg cgcaatgtta ttcgtccgac ccagcgttat gcagatatgc tggaatttag
     3481 ctatcatccg aattgcttta gcaatgccat tgaacgtgaa aaagtgctgc ataatatgtg
     3541 ggcctatccg tataaaaata aaaaagtggt gcattatgaa ttttcagatc tgattgatgg
     3601 cgatattccg attttttata ataatattag caaaaccagc ctgattgcca gtgatggttg
     3661 tctggttgaa gatttttatc aggaaagtgc cctgaatcgc tgcctgaata aaattaatga
     3721 tctgtgtgat gaagatatta gcattcagac cgtgtggctg gaaattgcac tgaatattta
     3781 taacccgtac aaatatatca atgatctgaa aaaccagaat agcaacaaat atatttatac
     3841 cggtctggaa ctgaatggca aaattattca ggcctgccag aaaattgaaa aaaaaatctt
     3901 taaacgtgcc atctttaaca aaaaaaccaa taccgtgaat tggattgata ttaaactgga
     3961 tcaggattgg aatgtgggca ttctgaataa taatatgtat gatggcctgc ctggtatttt
     4021 tattttttat gtggccctga aatatattac caaaaaccat aaatatgatt atgtgatcga
     4081 atgcattaaa aatagcattt ataccattcc gagcgaagat attctgagcg cattttttgg
     4141 taaaggcagc ctgatttatc cgctgctggt tgattatcgc ctgaataatg atattaatag
     4201 cctgaatgtg gccgtggaaa ttgccgatat gctgattgaa aaaaaaccga ttaataatgg
     4261 cgaactgaaa aatgattgga ttcatggcca taatagcatt attaaagtgc tgctgctgct
     4321 gagcgaaatt accgaagatg aaaaatatcg caaatttagc ctggaaattt ttgaaaaact
     4381 gagcgaagaa ccgtatttta attttcgtgg ttttggccat ggcatttata gctatgttca
     4441 tctgctgagc aaatttaatc gcattgataa agccaatagc ctgctgcata aaattaaaga
     4501 aagctatttt gaagaagaac cgaaaaataa ttcctggtgt aaaggcaccg ttggtgaact
     4561 gctggcaacc attgaactgt atgatgataa tattagtaac atcgatatta acaaaaccat
     4621 tgcctataaa aataaagatt gcctgtgcca tggcaatgca ggcaccctgg aaggtctgat
     4681 tcagctggca aaaaaagatc cggaaaccta tcagtataaa aaaaataaac tgatcagcta
     4741 tatgctgaat gattttgaaa aaaataatac cctgaaagtg gcaggcagcg aatatctgga
     4801 aagcctgggt ttttttgttg gtattagcgg tgttggttat gaactgctgc gtaatctgga
     4861 tagcgaaatt ccgaatgcac tgctgtttga actgtaaggt accctcgagt ctggtaaaga
     4921 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     4981 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     5041 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
     5101 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     5161 ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg
     5221 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5281 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     5341 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     5401 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     5461 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     5521 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     5581 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     5641 cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca
     5701 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     5761 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     5821 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     5881 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     5941 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     6001 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     6061 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa
     6121 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6181 tagaaaaata aacaaatagg catgcagcgc tcttccgctt cctcgctcac tgactcgcta
     6241 cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact caaaagcggt aatacggtta
     6301 tccacagaat caggggataa agccggaaag aacatgtgag caaaaagcaa agcaccggaa
     6361 gaagccaacg ccgcaggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6421 tcgacgctca agccagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6481 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6541 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt ggtatctcag
     6601 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6661 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6721 gccactggca gcagccattg gtaactgatt tagaggactt tgtcttgaag ttatgcacct
     6781 gttaaggcta aactgaaaga acagattttg gtgagtgcgg tcctccaacc cacttacctt
     6841 ggttcaaaga gttggtagct cagcgaacct tgagaaaacc accgttggta gcggtggttt
     6901 ttctttattt atgagatgat gaatcaatcg gtctatc
//
