LOCUS       sequence-438165-        5648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express ChmI in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5648)
  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 5648)
  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: pETDuet-1-chmI
FEATURES             Location/Qualifiers
     source          1..5648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        445..463
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    464..488
                     /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             597..641
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(679..697)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      693..740
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      777..1232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(864..883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1074..1095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1347..2138,2139..2207))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1970..1989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2208..2300)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2381..2969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2870..2889
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3123..3140
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3155..3294
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3220..3239)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3380..3402
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3396..3587)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4159..5241)
                     /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     5203..5222
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5242..5319)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5525..5544
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5632..5648,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgatggaaga
      121 cgtcaatgaa ctgtgcggcc tgatccaggc gatcatctcg gaatcccacg ggaccctgga
      181 cgagaaggtg gagcccgaca caccgctgct cgtatcgggg ctcctggact ccctgaccat
      241 catgcggatc gtggcgcggg tcgaggcgac caccgggatc tccttcccgg agacctcgct
      301 cgtggcggcc aacttccgta cgcccgactc cctgtgggcg gccatcggga agacccgcgc
      361 cgcctccggc gacgggagcc cggtgtcatg atgcttaagt cgaacagaaa gtaatcgtat
      421 tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata
      481 acaattcccc atcttagtat attagttaag tataagaagg agatatacat atggcagatc
      541 tcaattggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag
      601 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
      661 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
      721 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
      781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
      841 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      961 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1021 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1081 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1141 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1201 gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca
     1261 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     1321 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     1381 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1441 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1501 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1561 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1621 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1681 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1741 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1801 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1861 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1921 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1981 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     2041 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     2101 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     2161 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     2221 caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2281 tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2341 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2401 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2461 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2521 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2581 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2641 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2701 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2761 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2821 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2881 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2941 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3001 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3061 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3121 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3181 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3241 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3301 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3361 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3421 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3481 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3541 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3601 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3661 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3721 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3781 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3841 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3901 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3961 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4021 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     4081 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4141 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4201 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4261 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4321 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4381 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4441 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4501 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4561 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4621 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4681 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4741 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     4801 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     4861 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     4921 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     4981 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5041 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5101 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5161 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5221 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5281 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5341 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5401 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5461 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5521 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5581 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac
     5641 tcactata
//
