LOCUS       sequence-438168-        6477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express LflChmG in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6477)
  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 6477)
  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-LflChmG
FEATURES             Location/Qualifiers
     source          1..6477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1192..1209
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1262..1280)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1276..1323
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1360..1815
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1447..1466)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1657..1678
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1907..2722)
                     /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(1907..2722)
                     /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     2630..2649
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2714..2733
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2844..3432
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3333..3352
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3586..3603
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3618..3757
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3683..3702)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3843..3865
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3859..4050)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4859..5941)
                     /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     5903..5922
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5942..6019)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6225..6244
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6328..6347
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6328..6346
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6347..6371
                     /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             6386..6408
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6427..6444
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6454..6471
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 cggaccatcg acgtgcacga tatgggagta acgctgagac aagtagctga ggtagcagat
       61 gagctgcgcc tgctcgcggc tgaagtagac gatgggagag tcccagcgag ccgcgggtac
      121 gcggcagttc gaaatgcggg gttactgcgg ttgctgttgc cacaatccgc gggtgggtcg
      181 ggcttatcct acttagacta tactagagtg ctggaagagc tcgcactcgg ggacgcgagt
      241 gctgccttgg gatacaacat gcataacgtg gcaataggcg ggctctgcga atcggcagag
      301 cgcgacttgc caccctcagc tgcggctttc agacggtggg tgttcgacga ggtagctcat
      361 cgcgaccgca tgttcgctag tgctaccagt gaagtaggcg gtggcgctaa actcacagcg
      421 gtaaaagcta cttatcggcg gacagctgac gggttcgtgc tgaatggccg caagtcgttc
      481 gtttcattag ctggaattgc cgattactac ataaccacag ctcggcggga agactccgac
      541 gcagctgacg aggtctcaca cttcgttgtt gccaaggacg acgcaggggt tagtttcgac
      601 ggcttatggg atggtgcagc cctcgcagga accgcgacct cggccatgac cctttctaac
      661 gtgcaccttg gtaccgatag actgttcttg ggaatagaag gtatgagcct cttcaagctt
      721 gttcgggaac cgcactggat ggtctctggt tacatgggtg cctacctggg tgttgccgag
      781 tctattcttc gccacgtcac cacatttgtt accgccgacg caaagcgccg ggacagtgca
      841 gttgtgcgag ctgaggtagg tcgcatggcc gttgaccttg cagcgacacg cgccttagtt
      901 catgctgcgg ctagattggt ggatgagaga aaagggtccg ttgaagccaa ctcggccgta
      961 cacgcggcca aataccgcgt cggagaatct acgacaactc tggctcaagc cgcagttcgt
     1021 atttgcggga gcactgcctt gcgacgatct aatcccctgg agcgtctcct gcgcgaggcc
     1081 tcattttgca gcgtcatgcc cgcgaaacca gatgagtgct tagagtacgt tggtaaggcg
     1141 agacttggct tcaatatgtt cgactctacg actgtggaat ggtgactcga gcaccaccac
     1201 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1261 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1321 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1381 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1441 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1501 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1561 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1621 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1681 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1741 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1801 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1861 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     1921 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1981 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     2041 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     2101 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     2161 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     2221 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     2281 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     2341 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     2401 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     2461 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     2521 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     2581 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     2641 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     2701 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     2761 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2821 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2881 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2941 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3001 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3061 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3121 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3181 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3241 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3301 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3361 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3421 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3481 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3541 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3601 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3661 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3721 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3781 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3841 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3901 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3961 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     4021 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     4081 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     4141 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     4201 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4261 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4321 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4381 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4441 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4501 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     4561 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     4621 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     4681 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     4741 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4801 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4861 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4921 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     4981 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5041 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     5101 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     5161 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5221 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5281 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     5341 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     5401 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     5461 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5521 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5581 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     5641 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     5701 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     5761 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     5821 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     5881 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     5941 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     6001 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     6061 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6121 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     6181 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     6241 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     6301 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     6361 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc
     6421 agcagccatc atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatg
//
