LOCUS       sequence-438167-        6843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express ChmJ in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6843)
  AUTHORS   Crooke AM, Chand AK, Cui Z, Balskus EP
  TITLE     Elucidation of Chalkophomycin Biosynthesis Reveals 
            N-Hydroxypyrrole-Forming Enzymes.
  JOURNAL   J Am Chem Soc. 2024 Jun 12;146(23):16268-16280. doi: 
            10.1021/jacs.4c04712. Epub 2024 May 29.
  PUBMED    38810110
REFERENCE   2  (bases 1 to 6843)
  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/jacs.4c04712"; journalName: "J Am Chem Soc"; date: 
            "2024-06-12- 12"; volume: "146"; issue: "23"; pages: "16268-16280"
COMMENT     Sequence Label: pET28a-ChmJ
FEATURES             Location/Qualifiers
     source          1..6843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             11..28
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1592..1609
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1662..1680)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1676..1723
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1760..2215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1847..1866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2057..2078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2307..3122)
                     /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             complement(2307..3122)
                     /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     3030..3049
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3114..3133
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3244..3832
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3733..3752
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3986..4003
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4018..4157
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4083..4102)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4243..4265
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4259..4450)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5259..6341)
                     /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     6303..6322
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6342..6419)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6625..6644
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6728..6747
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6728..6746
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6747..6771
                     /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)."
     RBS             6786..6808
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(6827..6843,1)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 cagcagcggc ctggtgccgc gcggcagcca tatgagccgt ccccccgcgg caccccgcct
       61 cgaccggctc ctggcggacg cggcgcgcga ccatccggac cggcccgccg tcgaggcgga
      121 cggacggacc ctcacctacg cggaactgga gcggaccgtc ggcgaactgg ccggcgccct
      181 caccgccgcc ggggtccgcc ccggcgaccg cgtcgggatc catgtgccca agagcatcga
      241 ggccgtcctc ggcatctacg ctgtcctgcg gtccggcgcc gtcgccgccc cgctcgacat
      301 ggcggcgccc ccggagcgga ccgccaggat ggtgaagaac gcgggcatcg tcttcctcat
      361 cacctcggcg gcgaacccgg ccgccacccg ggcggtgaag gagtgcgccg acggcggcac
      421 ggcggacccc cggcccctcg ggtcccacct gggcatcctc cccgtcgact gcccggcggc
      481 cccgctcacc gacgcggccg gcgcggggta cgtgctcttc acctcgggca gcacgggctg
      541 gcccaagggg gtgctcctct cccacgagaa cgtcctccac ttcgtccggt ggaccgtcga
      601 gcggttcggg gtcacccccg ccgaccggat cggctcccag tcctcgctga ccttcgacct
      661 cagcaccttc gacctcttcg gcagcgcgct gtccggcgcc tgcctggtgc tgctgcccga
      721 cctgctcaag ggcttcccgc gcgatgtggt ggagtggctg cggaccgagc ggatatcggt
      781 gttctacgcc gtgccgacgc tgtaccagtc gatgctgcac aagggcgggg ccaccggcgc
      841 ggaactgccc ggcctgcgca tcatcgcctt cgccggggag cccttccccg ccgccgaact
      901 ccagcggtac accgagctgt tcgggagcgc cgagttctac aacctctacg gccccaccga
      961 gaccaacgtc tgcaccttcg agcgggtgga ccaccggtgg gaccctcggg acggcctctc
     1021 catcggccgg gctatcgacg gcgtccgcgt cgaactcgtc gacgaggcgc accgggccac
     1081 ccgggacgag ggcgagatcg ccgtgtcggg cccgacggtc ttcctcggct acctcaccgg
     1141 cggtgagctg cgcccgctca ccacccggat ctcccccgac gacggctccc ccgcctatct
     1201 caccggcgac ctcggccact acggagccga cggaaagatc tatctgcgcg gacgccgcga
     1261 ccaccaggtg aagcgccggg gataccgcat cgacctgttc gacatcgaga gcgtcgtgca
     1321 ggagttcccg gcggtacgca cctgtgcggc ggtgtggacg gagcccgccc cgggcgaggg
     1381 acgcatcgtc ctctacgcgg tcgccgacgg catgacgcag acgctgctgc gccgtgccgt
     1441 atcggcggca ctgccgcggc acatggtgcc cgacgacatc catctcgtcg gccggctggc
     1501 actcaccggc cggggaaaga tcgaccgcga agcgctggcc tcggcggcgc cgacccagga
     1561 aagggatcat ggaagacgtc aatgactcga gcaccaccac caccaccact gagatccggc
     1621 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1681 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1741 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1801 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1861 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1921 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1981 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2041 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2101 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2161 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     2221 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2281 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     2341 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     2401 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     2461 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     2521 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     2581 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     2641 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     2701 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2761 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     2821 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2881 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2941 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     3001 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     3061 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     3121 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     3181 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3241 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3301 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3361 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3421 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3481 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3541 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3601 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3661 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3721 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3781 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3841 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3901 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3961 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     4021 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     4081 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     4141 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4201 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4261 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     4321 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     4381 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     4441 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     4501 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     4561 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4621 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4681 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4741 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4801 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4861 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     4921 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4981 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     5041 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     5101 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     5161 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     5221 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5281 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5341 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     5401 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5461 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     5521 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     5581 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5641 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5701 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5761 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5821 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5881 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5941 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     6001 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6061 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     6121 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     6181 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     6241 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     6301 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     6361 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     6421 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     6481 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     6541 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6601 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6661 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     6721 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     6781 ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca
     6841 tca
//
