LOCUS       sequence-449514-        4183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses precursor peptide ChrA-C63S in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4183)
  AUTHORS   Ayikpoe RS, Zhu L, Chen JY, Ting CP, van der Donk WA
  TITLE     Macrocyclization and Backbone Rearrangement During RiPP Biosynthesis
            by a SAM-Dependent Domain-of-Unknown-Function 692.
  JOURNAL   ACS Cent Sci. 2023 Apr 24;9(5):1008-1018. doi: 
            10.1021/acscentsci.3c00160. eCollection 2023 May 24.
  PUBMED    37252350
REFERENCE   2  (bases 1 to 4183)
  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/acscentsci.3c00160"; journalName: "ACS Cent Sci"; date: 
            "2023-04-24- 24"; volume: "9"; issue: "5"; pages: "1008-1018"
COMMENT     Sequence Label: pACYCDuet-1:ChrA-C63S
FEATURES             Location/Qualifiers
     source          1..4183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        387..405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    406..430
                     /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             539..583
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(621..639)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      635..682
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(903..1562)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1477..1496
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1563..1665)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2057..2074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2191..2736)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2271..2290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2961..4043)
                     /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     4005..4024
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4044..4121)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4167..4183,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccagatga agattccggc
      121 tctggtgatg gccagcctgc tggcggtatc agtgagcggg caaaccacga aaccggtcaa
      181 aaaaggcacg aaaagtgtta aaaaagtgaa aaaaatggat agcccaaaaa ccgttaaagc
      241 agaacccacc aaagtcgtta aacgtgacac gatcctgaaa catggcggcg gttctccggc
      301 gtgcggtatg gggtgaagct tgcggccgca taatgcttaa gtcgaacaga aagtaatcgt
      361 attgtacacg gccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga
      421 taacaattcc ccatcttagt atattagtta agtataagaa ggagatatac atatggcaga
      481 tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg agtctggtaa
      541 agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta
      601 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
      661 acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact
      721 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
      781 ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt catccgctta
      841 ttatcactta ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa
      901 aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
      961 acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     1021 tcgccttgcg tataatattt gcccatagtg aaaacggggg cgaagaagtt gtccatattg
     1081 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata
     1141 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     1201 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     1261 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     1321 tcaccgtctt tcattgccat acggaactcc ggatgagcat tcatcaggcg ggcaagaatg
     1381 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     1441 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     1501 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     1561 attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat
     1621 cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc
     1681 caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg
     1741 aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac
     1801 ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag
     1861 ctgtccctcc tgttcagcta ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca
     1921 tcagcgctag cggagtgtat actggcttac tatgttggca ctgatgaggg tgtcagtgaa
     1981 gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg tcagcagaat atgtgataca
     2041 ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag
     2101 cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca
     2161 gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca
     2221 tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca
     2281 ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg
     2341 tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt
     2401 aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg
     2461 ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg
     2521 cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc
     2581 taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa
     2641 gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca
     2701 gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata
     2761 aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc
     2821 atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga
     2881 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     2941 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3001 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     3061 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     3121 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     3181 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     3241 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     3301 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     3361 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     3421 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     3481 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     3541 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     3601 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     3661 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     3721 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     3781 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     3841 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     3901 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     3961 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     4021 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     4081 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     4141 cttatgcgac tcctgcatta ggaaattaat acgactcact ata
//
