LOCUS       sequence-438171-        6921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express ChmK in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6921)
  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 6921)
  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-ChmK
FEATURES             Location/Qualifiers
     source          1..6921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(57..75)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      71..118
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      155..610
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(242..261)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     452..473
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(702..1517)
                     /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(702..1517)
                     /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     1425..1444
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1509..1528
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1639..2227
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2128..2147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2381..2398
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2413..2552
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2478..2497)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2638..2660
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2654..2845)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3654..4736)
                     /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     4698..4717
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4737..4814)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5020..5039
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5123..5142
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5123..5141
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5142..5166
                     /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             5181..5203
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5222..5239
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5249..5266
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(6908..6921,1..4)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
       61 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
      121 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
      181 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
      241 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
      301 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
      361 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
      421 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
      481 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
      541 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
      601 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      661 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
      721 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
      781 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
      841 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
      901 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
      961 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     1021 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     1081 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     1141 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     1201 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     1261 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1321 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     1381 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     1441 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     1501 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     1561 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1621 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1681 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1741 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     1801 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1861 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1921 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1981 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2041 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2101 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2161 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2221 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     2281 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     2341 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     2401 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2461 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     2521 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     2581 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     2641 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     2701 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     2761 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     2821 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     2881 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     2941 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     3001 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     3061 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     3121 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     3181 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     3241 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     3301 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     3361 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     3421 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     3481 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     3541 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     3601 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     3661 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3721 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     3781 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     3841 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     3901 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     3961 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     4021 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     4081 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     4141 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     4201 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     4261 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     4321 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     4381 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     4441 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     4501 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     4561 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     4621 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     4681 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     4741 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     4801 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     4861 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     4921 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     4981 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     5041 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     5101 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     5161 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag
     5221 ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata tgaccgccga
     5281 tgtggttggc tggccggcca cggatgccga gaaatatcgg aacgccggat actggcgagg
     5341 ggagtccttc gcccagttcc tgagcgcccg gacccgggcg tacggcgacc ggaccgccgt
     5401 cgtcgacgac cggacccggc tgagctacgc cgagttggac gagcagtccg accgggtggc
     5461 acgggggctc gcggacctcg ggatcgccgc cggtgaccgg gtgctggtcc agctccccaa
     5521 ccgggtggag ttcctgctgc tctggttcgc gctgatgaag accggcgcgg tgccggtcca
     5581 cacccagccg ggccaccgca gcagcgagat cacccacctg gcccgcctct ccggggcggt
     5641 ggcctacgtc atccccgaca agcacgcgga cttcgaccac cggctgctgg cccaggccgt
     5701 acggaacgac agcccggccc tcaagcacgt ggtcgtggtc ggcgacccgg gacacggcac
     5761 cggattcacc tccttcgcgg acctgttgac ggaccggccg gccgccagga cgccgaagtc
     5821 ccgtgcctgg ggcgaggaca tggcgctcct gctgctctcc ggaggcacca ccgggctgcc
     5881 caagctcatc ccccgcaccc acgacgacta cctctacaac gggcgtgccg cggccgaggt
     5941 ctgcgagctg accagccggt ccgtctatct cgcggcgctg ccggtggcgt tcaactacac
     6001 gatgaactgc ccgggtgtgc tggggaccct cggcgtcggc gggaaggtcg tcctggcgag
     6061 caacccggac ccggactact gcttcggcct gatcgggcgc gagggcgtca ccatcgccgc
     6121 catcaacccg cagatggcgc ccgtctggct ggacgaggtg tcgatgaccg acgccgacct
     6181 gtccaccctg cgggtcctcc agatcggcag cgcgcggctg gccgacgagc aggccgagcg
     6241 gatgaccgcc gggttcggct gcacgatcca gcaggtgttc gggatggccg agggcctgct
     6301 ctgcctgacc cggctcaccg accctccgga gatcatcaac tcggtccagg gccggccgat
     6361 ctcgcctgcc gacgaggtac gggtggtggg accggacgac cgggaggtgg gcgagggcga
     6421 ggagggcgaa ctcctcaccc gcggcccgta caccctgcgc ggctactacc gggcggcgga
     6481 gcacaacgcc aggagcttca ccccggacgg cttctaccgc agcggcgatc tcgtacgccg
     6541 gctgccgtcc ggacacctca tcgtggtggg gcggtcgaag gaccagatca accggggcgg
     6601 cgagaagatc ccggccaccg aggtcgaggg ccacctgctg gcccaccccg ggatccggtc
     6661 ggtcgccctg gtggccgcgc ccgaccccga cttcggcgag cggcccgtcg ccttcgtcgt
     6721 gccggccggc gggccggccc cggaccgcag ggacctcgcg gccttcctcg ccgagagggg
     6781 actggccgcc tacaaggcgc ccgccgaggt gcggacgatc gacgcgatgc cgctgacccc
     6841 cgtcggaaag atcgacaaga aggtgctggc cgcctcgctc cgggaggggt cggccggatg
     6901 actcgagcac caccaccacc a
//
