LOCUS       sequence-458632-        4183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses precursor peptide ChrA 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
FEATURES             Location/Qualifiers
     source          1..4183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     110..129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     326..343
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        735..837
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             838..1497
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(904..923)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(1718..1765)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1761..1779
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1817..1861)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    1970..1994
                     /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(1995..2013)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2300..2317)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    2373..2397
                     /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(2398..2416)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2462..2539
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2540..3622
                     /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(2559..2578)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      join(3847..4183,1..209)
                     /direction=RIGHT
                     /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.
                     "
ORIGIN
        1 cccggaactg agtgtcaggc gtggaatgag acaaacgcgg ccataacagc ggaatgacac
       61 cggtaaaccg aaaggcagga acaggagagc gcacgaggga gccgccaggg ggaaacgcct
      121 ggtatcttta tagtcctgtc gggtttcgcc accactgatt tgagcgtcag atttcgtgat
      181 gcttgtcagg ggggcggagc ctatggaaaa acggctttgc cgcggccctc tcacttccct
      241 gttaagtatc ttcctggcat cttccaggaa atctccgccc cgttcgtaag ccatttccgc
      301 tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg aatatatcct
      361 gtatcacata ttctgctgac gcaccggtgc agcctttttt ctcctgccac atgaagcact
      421 tcactgacac cctcatcagt gccaacatag taagccagta tacactccgc tagcgctgat
      481 gtccggcggt gcttttgccg ttacgcacca ccccgtcagt agctgaacag gagggacagc
      541 tgatagaaac agaagccact ggagcacctc aaaaacacca tcatacacta aatcagtaag
      601 ttggcagcat cacccgacgc actttgcgcc gaataaatac ctgtgacgga agatcacttc
      661 gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg
      721 cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga
      781 tcactaccgg gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg
      841 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
      901 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
      961 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     1021 attcttgccc gcctgatgaa tgctcatccg gagttccgta tggcaatgaa agacggtgag
     1081 ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg
     1141 ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg
     1201 caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat
     1261 atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc
     1321 aatatggaca acttcttcgc ccccgttttc actatgggca aatattatac gcaaggcgac
     1381 aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc
     1441 ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt
     1501 ttttaaggca gttattggtg cccttaaacg cctggttgct acgcctgaat aagtgataat
     1561 aagcggatga atggcagaaa ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg
     1621 gtcgttaaat agccgcttat gtctattgct ggtttaccgg tttattgact accggaagca
     1681 gtgtgaccgt gtgcttctca aatgcctgag gtttcagcaa aaaacccctc aagacccgtt
     1741 tagaggcccc aaggggttat gctagttatt gctcagcggt ggcagcagcc taggttaatt
     1801 aagctgcgct agtagacgag tccatgtgct ggcgttcaaa tttcgcagca gcggtttctt
     1861 taccagactc gagggtaccg acgtcagcga tcgcgtggcc ggccgatatc caattgagat
     1921 ctgccatatg tatatctcct tcttatactt aactaatata ctaagatggg gaattgttat
     1981 ccgctcacaa ttcccctata gtgagtcgta ttaatttcga ttatgcggcc gtgtacaata
     2041 cgattacttt ctgttcgact taagcattat gcggccgcaa gcttcacccc ataccgcacg
     2101 ccggacaacc gccgccatgt ttcaggatcg tgtcacgttt aacgactttg gtgggttctg
     2161 ctttaacggt ttttgggcta tccatttttt tcactttttt aacacttttc gtgccttttt
     2221 tgaccggttt cgtggtttgc ccgctcactg ataccgccag caggctggcc atcaccagag
     2281 ccggaatctt catctggctg tggtgatgat ggtgatggct gctgcccatg gtatatctcc
     2341 ttattaaagt taaacaaaat tatttctaca ggggaattgt tatccgctca caattcccct
     2401 atagtgagtc gtattaattt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     2461 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     2521 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     2581 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     2641 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     2701 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     2761 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     2821 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     2881 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     2941 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     3001 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     3061 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     3121 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     3181 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     3241 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     3301 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga
     3361 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     3421 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     3481 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     3541 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     3601 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     3661 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     3721 cgcggggcat gactaacatg agaattacaa cttatatcgt atggggctga cttcaggtgc
     3781 tacatttgaa gagataaatt gcactgaaat ctagaaatat tttatctgat taataagatg
     3841 atcttcttga gatcgttttg gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc
     3901 cttgcagggc ggtttttcga aggttctctg agctaccaac tctttgaacc gaggtaactg
     3961 gcttggagga gcgcagtcac caaaacttgt cctttcagtt tagccttaac cggcgcatga
     4021 cttcaagact aactcctcta aatcaattac cagtggctgc tgccagtggt gcttttgcat
     4081 gtctttccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcggactgaa
     4141 cggggggttc gtgcatacag tccagcttgg agcgaactgc cta
//
