LOCUS       sequence-438170-        6081 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express ChmF in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6081)
  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 6081)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-ChmF
FEATURES             Location/Qualifiers
     source          1..6081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(54..72)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      68..115
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      152..607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(239..258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     449..470
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(699..1514)
                     /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(699..1514)
                     /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     1422..1441
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1506..1525
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1636..2224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2125..2144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2378..2395
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2410..2549
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2475..2494)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2635..2657
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2651..2842)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3651..4733)
                     /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     4695..4714
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4734..4811)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5017..5036
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5120..5139
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5120..5138
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5139..5163
                     /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             5178..5200
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5219..5236
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5246..5263
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
       61 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
      121 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
      181 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      241 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      301 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
      361 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
      421 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
      481 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
      541 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
      601 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
      661 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
      721 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
      781 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
      841 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
      901 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
      961 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     1021 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     1081 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     1141 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     1201 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     1261 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     1321 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     1381 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     1441 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     1501 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     1561 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1621 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1681 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1741 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1801 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1861 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1921 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1981 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2041 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2101 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2161 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2221 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2281 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2341 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2401 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     2461 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     2521 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     2581 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     2641 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     2701 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     2761 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     2821 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     2881 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     2941 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     3001 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     3061 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     3121 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     3181 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     3241 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     3301 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     3361 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     3421 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     3481 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     3541 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     3601 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     3661 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3721 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     3781 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     3841 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     3901 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     3961 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4021 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     4081 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4141 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     4201 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     4261 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     4321 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     4381 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     4441 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     4501 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     4561 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     4621 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     4681 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     4741 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     4801 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     4861 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     4921 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     4981 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     5041 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     5101 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     5161 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca
     5221 tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgc cggagatcga
     5281 gctgaagcgc ggccccgtcg agtaccggcg gatagcgggg cgggacaacc tcgccccact
     5341 ggtgttcctg cacgaagggc tcggctgcgt cgcgatgtgg ggccggttcc ccgacgccct
     5401 cgcgcgggcg agcggccggc ccgcactggt ctactcccgc cacggatacg gcggttcggg
     5461 cccggcacgc acgccgcgcc cggtcaccta cctgcgggag gaggccaccg acgtcctgcc
     5521 ggagctcctg gaccgcctcg gcctggaacg ccccgtcctc gtcggacaca gtgacggggc
     5581 ctccatcgcc ctgctgcacg cgggcgtgcg tccggtgtcg gccctggtgc tcatcgcgcc
     5641 gcacgtcttc gtcgaaccgg agaccctggc cgggatcgtg gccgcccggg ccgcctaccg
     5701 ggacggtccg ctggcgggcc ggctggccgg attccacgac gacccggaca ccgccttcca
     5761 ctcctgggcg gacgtctggc tctcccccgc gttccgcgac tggaacatcg aggacgagct
     5821 gcacggggtc gaggcccccg tcctgctgat ccagggcgaa cgcgaccagt acgggaccgt
     5881 ccgtcagctg cacgccgtcg agcggggcgt cgccgggccc gcccgcctgg tcgaaccggc
     5941 gggctgcggg cactcccccc atctggagcg gcccgccgag accctggagg cggtggccgg
     6001 cttcctcgcg gacgaggtga cggcggcgcg gcgcacccgc gccccggcgc cgacgtcctg
     6061 actcgagcac caccaccacc a
//
