LOCUS       sequence-438175-        6033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express C47A ChmN in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6033)
  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 6033)
  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-ChmN-C47A
FEATURES             Location/Qualifiers
     source          1..6033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(74..92)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      88..135
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      172..627
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(259..278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     469..490
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(719..1534)
                     /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(719..1534)
                     /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     1442..1461
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1526..1545
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1656..2244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2145..2164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2398..2415
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2430..2569
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2495..2514)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2655..2677
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2671..2862)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3671..4753)
                     /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     4715..4734
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4754..4831)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5037..5056
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5140..5159
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5140..5158
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5159..5183
                     /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             5198..5220
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
       61 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      121 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      181 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      241 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      301 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      361 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
      421 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
      481 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
      541 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
      601 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
      661 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
      721 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
      781 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
      841 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
      901 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
      961 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     1021 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1081 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1141 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1201 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1261 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1321 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1381 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     1441 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     1501 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     1561 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     1621 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1681 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1741 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1801 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1861 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1921 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1981 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2041 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2101 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2161 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2221 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2281 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2341 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2401 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2461 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2521 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     2581 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2641 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     2701 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     2761 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     2821 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     2881 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     2941 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3001 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3061 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3121 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3181 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3241 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     3301 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     3361 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     3421 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     3481 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     3541 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     3601 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     3661 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3721 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     3781 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     3841 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     3901 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     3961 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4021 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4081 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4141 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4201 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4261 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4321 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4381 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4441 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4501 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4561 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     4621 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     4681 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     4741 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     4801 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     4861 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     4921 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     4981 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5041 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     5101 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     5161 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
     5221 tataccatgc gtatgttgtc ggcgaacacc ctgccgtccc ccgggagtct cacctcccag
     5281 gtgataggtg aaattcccgc atggggaatg tccagcgcga cggaactgat ggggaaactg
     5341 acctcctcca ccgcgccgtt ccccgccacc ttcgcggtct ccgcggcgaa gaaggcctcg
     5401 ctgcggttcg gcttcatcga tgacctcgac gaccaggaga cctgggcggc gttacccggg
     5461 atcatcacgt cctacctgga gacctaccag cagatcgccc gggagacctc cctcatcgtg
     5521 ctcttccgcc ccgagcggga gaccggcgac gtcgcgcact accaccggaa gttctgggac
     5581 gtcctgcagt acctgcaccg gcacgacccc gagtcgtggc cggacggcat ccccgaggac
     5641 cccgaggacg gccgctggga gttcagcttc gccggcaccc ccatcttcgt cgtctgcaac
     5701 acccccgcgc acaagcgccg ggcgagccgg tcgaaccccg aattcatgat caccttccag
     5761 ccgcgctggg tgttcgagga gctggactcc acctcggcgc ggggcatcgc ggcccggcgt
     5821 gtcatccgca agcggctgcg cgccttcgac agcgaacagc cctcgccgga gctggggagc
     5881 tacggagatc cggagaaccg ggaatggcgt cagtacttcc tccccgacga caacgcggag
     5941 aagcccggtc gctgcccctt ccgccacggc gcgcacggcg ccacccccgc ggcacagggt
     6001 gagggcaccg gtgacaagct tgcggccgca ctc
//
