LOCUS       sequence-458633-        4183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses precursor peptide ChrA-C63S/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-C63S-C66S
FEATURES             Location/Qualifiers
     source          1..4183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     529..548
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(615..717)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1109..1126)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1243..1788)
                     /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(1323..1342)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2013..3095)
                     /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     3057..3076
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3096..3173)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        3219..3237
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3238..3262
                     /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             3318..3335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3622..3640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3641..3665
                     /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             3774..3818
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3856..3874)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3870..3917
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(join(4138..4183,1..614))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc
       61 atcagcacct tgtcgccttg cgtataatat ttgcccatag tgaaaacggg ggcgaagaag
      121 ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag
      181 acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac
      241 gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag
      301 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
      361 catatcacca gctcaccgtc tttcattgcc atacggaact ccggatgagc attcatcagg
      421 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt
      481 aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga
      541 aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg
      601 atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg
      661 cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg
      721 tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt
      781 atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt
      841 gatgctgcca acttactgat ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc
      901 tgtttctatc agctgtccct cctgttcagc tactgacggg gtggtgcgta acggcaaaag
      961 caccgccgga catcagcgct agcggagtgt atactggctt actatgttgg cactgatgag
     1021 ggtgtcagtg aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga
     1081 atatgtgata caggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc
     1141 gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga
     1201 agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc
     1261 ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact
     1321 ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt
     1381 tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac
     1441 tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt
     1501 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa
     1561 aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc
     1621 gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta
     1681 cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt
     1741 ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt
     1801 attaatcaga taaaatattt ctagatttca gtgcaattta tctcttcaaa tgtagcacct
     1861 gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc
     1921 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     1981 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     2041 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2101 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     2161 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     2221 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     2281 cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     2341 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     2401 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     2461 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     2521 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     2581 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     2641 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     2701 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     2761 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     2821 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     2881 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     2941 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     3001 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     3061 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     3121 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     3181 tcgacgctct cccttatgcg actcctgcat taggaaatta atacgactca ctatagggga
     3241 attgtgagcg gataacaatt cccctgtaga aataattttg tttaacttta ataaggagat
     3301 ataccatggg cagcagccat caccatcatc accacagcca gatgaagatt ccggctctgg
     3361 tgatggccag cctgctggcg gtatcagtga gcgggcaaac cacgaaaccg gtcaaaaaag
     3421 gcacgaaaag tgttaaaaaa gtgaaaaaaa tggatagccc aaaaaccgtt aaagcagaac
     3481 ccaccaaagt cgttaaacgt gacacgatcc tgaaacatgg cggcggttct ccggcgtccg
     3541 gtatggggtg aagcttgcgg ccgcataatg cttaagtcga acagaaagta atcgtattgt
     3601 acacggccgc ataatcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     3661 attccccatc ttagtatatt agttaagtat aagaaggaga tatacatatg gcagatctca
     3721 attggatatc ggccggccac gcgatcgctg acgtcggtac cctcgagtct ggtaaagaaa
     3781 ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa
     3841 cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     3901 tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc
     3961 cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc
     4021 ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg ccattcatcc gcttattatc
     4081 acttattcag gcgtagcaac caggcgttta agggcaccaa taactgcctt aaaaaaatta
     4141 cgccccgccc tgccactcat cgcagtactg ttgtaattca tta
//
