LOCUS       sequence-455755-        4183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses precursor peptide ChrA-C66S 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-C66S
FEATURES             Location/Qualifiers
     source          1..4183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     522..541
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(608..710)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1102..1119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1236..1781)
                     /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(1316..1335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2006..3088)
                     /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     3050..3069
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3089..3166)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        3212..3230
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3231..3255
                     /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             3311..3328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3615..3633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3634..3658
                     /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             3767..3811
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3849..3867)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3863..3910
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(join(4131..4183,1..607))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca
       61 ccttgtcgcc ttgcgtataa tatttgccca tagtgaaaac gggggcgaag aagttgtcca
      121 tattggccac gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa
      181 acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat
      241 cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg
      301 aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca
      361 ccagctcacc gtctttcatt gccatacgga actccggatg agcattcatc aggcgggcaa
      421 gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg
      481 ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct
      541 caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt
      601 tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
      661 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt
      721 ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg gacaccagga tttatttatt
      781 ctgcgaagtg atcttccgtc acaggtattt attcggcgca aagtgcgtcg ggtgatgctg
      841 ccaacttact gatttagtgt atgatggtgt ttttgaggtg ctccagtggc ttctgtttct
      901 atcagctgtc cctcctgttc agctactgac ggggtggtgc gtaacggcaa aagcaccgcc
      961 ggacatcagc gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca
     1021 gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg
     1081 atacaggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg
     1141 gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact
     1201 taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac
     1261 aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga
     1321 taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt
     1381 accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc
     1441 cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg
     1501 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc
     1561 actggcagca gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt
     1621 aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt
     1681 tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt
     1741 tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc
     1801 agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca
     1861 gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga
     1921 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     1981 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2041 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     2101 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     2161 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     2221 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     2281 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     2341 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     2401 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     2461 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     2521 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     2581 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     2641 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     2701 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     2761 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     2821 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     2881 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     2941 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     3001 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     3061 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     3121 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     3181 tctcccttat gcgactcctg cattaggaaa ttaatacgac tcactatagg ggaattgtga
     3241 gcggataaca attcccctgt agaaataatt ttgtttaact ttaataagga gatataccat
     3301 gggcagcagc catcaccatc atcaccacag ccagatgaag attccggctc tggtgatggc
     3361 cagcctgctg gcggtatcag tgagcgggca aaccacgaaa ccggtcaaaa aaggcacgaa
     3421 aagtgttaaa aaagtgaaaa aaatggatag cccaaaaacc gttaaagcag aacccaccaa
     3481 agtcgttaaa cgtgacacga tcctgaaaca tggcggcggt tgtccggcgt ccggtatggg
     3541 gtgaagcttg cggccgcata atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc
     3601 cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     3661 atcttagtat attagttaag tataagaagg agatatacat atggcagatc tcaattggat
     3721 atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag aaaccgctgc
     3781 tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc
     3841 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     3901 gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt
     3961 caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc
     4021 gacgaccggg tcgaatttgc tttcgaattt ctgccattca tccgcttatt atcacttatt
     4081 caggcgtagc aaccaggcgt ttaagggcac caataactgc cttaaaaaaa ttacgccccg
     4141 ccctgccact catcgcagta ctgttgtaat tcattaagca ttc
//
