LOCUS       sequence-487216-        4960 bp    DNA     circular SYN 26-SEP-2025
DEFINITION  For expression of the Enterococcal cytolysin activating protease 
            CylA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4960)
  TITLE     Combatting Virulent Gut Bacteria by Inhibiting the Biosynthesis of a
            Two-component Lanthipeptide Toxin
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4960)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 26, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pRSF:His6-CylA-27-412
FEATURES             Location/Qualifiers
     source          1..4960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..750
                     /direction=RIGHT
                     /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     481..500
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     723..740
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        766..857
                     /gene="bla"
                     /label=AmpR promoter
     CDS             858..1673
                     /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             858..1673
                     /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     complement(931..950)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(1903..1950)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1946..1964
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2002..2046)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    2155..2179
                     /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)."
     promoter        complement(2180..2198)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(3443..3460)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    3516..3540
                     /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)."
     promoter        complement(3541..3559)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3605..3682
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3683..4765
                     /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     complement(3702..3721)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aacggaatag ctgttcgttg acttgataga ccgattgatt catcatctca taaataaaga
       61 aaaaccaccg ctaccaacgg tggttttctc aaggttcgct gagctaccaa ctctttgaac
      121 caaggtaagt gggttggagg accgcactca ccaaaatctg ttctttcagt ttagccttaa
      181 caggtgcata acttcaagac aaagtcctct aaatcagtta ccaatggctg ctgccagtgg
      241 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      301 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      361 actgagatac caacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      421 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
      481 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctggc ttgagcgtcg
      541 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgcctgcg gcgttggctt
      601 cttccggtgc tttgcttttt gctcacatgt tctttccggc tttatcccct gattctgtgg
      661 ataaccgtat taccgctttt gagtgagctg acaccgctcg ccgcagtcga acgaccgagc
      721 gtagcgagtc agtgagcgag gaagcggaag agcgctgcat gcctatttgt ttatttttct
      781 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
      841 attgaaaaag gaagagtatg agccatattc aacgggaaac gtcttgctct aggccgcgat
      901 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
      961 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     1021 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     1081 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     1141 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
     1201 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     1261 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     1321 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     1381 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     1441 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     1501 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     1561 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     1621 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa
     1681 ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1741 acatttcccc gaaaagtgcc acttgcggag acccggtcgt cagcttgtcg tcggttcagg
     1801 gcagggtcgt taaatagccg cttatgtcta ttgctggttt accggtttat tgactaccgg
     1861 aagcagtgtg accgtgtgct tctcaaatgc ctgaggtttc agcaaaaaac ccctcaagac
     1921 ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag cagcctaggt
     1981 taattaagct gcgctagtag acgagtccat gtgctggcgt tcaaatttcg cagcagcggt
     2041 ttctttacca gactcgaggg taccgacgtc agcgatcgcg tggccggccg atatccaatt
     2101 gagatctgcc atatgtatat ctccttctta tacttaacta atatactaag atggggaatt
     2161 gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgattatg cggccgtgta
     2221 caatacgatt actttctgtt cgacttaagc attatgcggc cgcttattta atgcgcactt
     2281 ctttgtattt cagcatgctg tttttatcga tgaagctatc ggcattttca aacagggtgt
     2341 tcagaacttc attgctgtct ttgctgttat ccacattttt gctcataatt gctgccagtg
     2401 ctgcgctaac ttccggtgtt gccaggctgg tgccaaagct cagggtataa ccatccggaa
     2461 aatctgctgc tttacccagc ggactaacca ggctggtcgg ataataggtc atcatcattt
     2521 cacgggcatc aatctgaccg gtgattttgt aattatcacc ataaccgcct gccggaccat
     2581 aaatgctcac gttgctacca taattgctgt aatcggcaat atcaccgctt tttttggttg
     2641 caccaacggt aataacgctt tccagaccac ccggaatgtg tttttcgtta ccggtgctaa
     2701 tatcacggct ttcattacct gcgcttgcaa caatcaggat gttatttttg cgtgcgtaat
     2761 taacaacttt acgaaaggct tcaacggtaa agcgttcatc atcaatttcc atgtttttgt
     2821 agctgcccag gctaacattg ataatatcca cctgatcatt ggtggcatcc acaattgctt
     2881 tcagcatatt aatgctattg ccatcggtgc catccataac tttatagctg ttcagattaa
     2941 cacgcggtgc aatggtatca ataacaccgg caacctgggt gccatgacca tattcatcca
     3001 gttcgatgtc gttcacatag tttttcagac gcagattatt gtcctgcaga ttcggatgca
     3061 gacgatcaat accgctatca atcagggcaa tggtaatatc tttacccagc tgttctttca
     3121 ccggatcaat atggctcagg atttttttgt acggccaatt aaagcttttg aaatctttat
     3181 tcaggatgtt aatgttcagg atgctgctat tcacttcttc cggtttaaaa tccggcagtt
     3241 ttttaccttc gctcacattg aaatatttgc cgatgaattt gcgatcgcta tcatccaggt
     3301 ttttgaagct caccagacca atttcttgaa tgtaatccac tttctcgctg ctcagttcgc
     3361 tttcaatttc ttcgatggcg gtggtctggc tattatcaat aaagaagctg atattgttgc
     3421 tcagcgaatt cggatcctgg ctgtggtgat gatggtgatg gctgctgccc atggtatatc
     3481 tccttattaa agttaaacaa aattatttct acaggggaat tgttatccgc tcacaattcc
     3541 cctatagtga gtcgtattaa tttcctaatg caggagtcgc ataagggaga gcgtcgagat
     3601 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     3661 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     3721 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     3781 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     3841 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     3901 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     3961 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     4021 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     4081 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     4141 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     4201 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     4261 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     4321 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     4381 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     4441 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata
     4501 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     4561 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     4621 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     4681 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     4741 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     4801 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     4861 gggcgcgggg catgactaac atgagaatta caacttatat cgtatggggc tgacttcagg
     4921 tgctacattt gaagagataa attgcactga aatctagagt
//
