LOCUS       sequence-438169-        6411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express LflChmH in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6411)
  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 6411)
  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-LflChmH
FEATURES             Location/Qualifiers
     source          1..6411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1126..1143
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1196..1214)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1210..1257
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1294..1749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1381..1400)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1591..1612
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1841..2656)
                     /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(1841..2656)
                     /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     2564..2583
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2648..2667
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2778..3366
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3267..3286
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3520..3537
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3552..3691
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3617..3636)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3777..3799
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3793..3984)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4793..5875)
                     /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     5837..5856
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5876..5953)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6159..6178
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6262..6281
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6262..6280
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6281..6305
                     /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             6320..6342
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6361..6378
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6388..6405
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 aacacgggtg cgactgattt cacggctcga tgggcaggca tagcgagttc tgcgttggcc
       61 ggtctcagca cagctgactt ccgaacacgg tggaaggctt tagcggactc aggtgttctc
      121 gctggagttc gcgacggacc gcgcggggta gtgaccgaca ccttagccgc aattgaaggg
      181 ctgggaatag gcggaacgtc tcctggtctt tgctacgcgt tgacatcgca gcatttcggg
      241 gttcagtctc ctctccgtgc cgccggatca gcagcttggg cggccttaag tgaaggtgtg
      301 gagcgaggag ataccttgat gtgtcatgcc ctgacagaag agggcggcgg gtcggaccca
      361 ttgtcaatgg caacacgggc ggaaccggca aacggtggct gggtcctgac cggtagaaaa
      421 gccttcgtga ctgcagctcc tatcgcggac cacgcgttgg ttttcgctcg aacggatgtt
      481 gaacgaactc cattcgccct tagcgcattc ttagtagacc tctctagcgc cggcgttagc
      541 cgttcagaac cgtttccaaa gaaggcgttg accgatgtcc cgatgggagc catcgatttt
      601 gactctgttc gtctgggtcc agagcagctc gtgggtcgtg agggtgctgg gctcgcctta
      661 ctgacaacaa caacttcttg ggagcggggc ttgctgttgg gttatgcact cggccctatg
      721 cgcctcgttt tagaccgtac cgttgagtgg gcccgccggc gaaagcaatt cgggcggcct
      781 atgggtgcct ctcacttagt cgcaggtcgc ctggcggaca tggcgacact gttacataga
      841 agtcgtacct taattttcgc catggcacaa cgcttcgact caggagaacc ggctcggcgt
      901 cttgcgtccg aagccgcaat gactaaggtc tccgtctcag aggacctctt atctttcaca
      961 cagcatgcgg cagcgcttgg cggtgtacga agctttgtca gtgataccgg tttaaccgca
     1021 gatctgacgg acgcgatggc cgctgcggtc tacgccggtc cgaatgacct tcttcgtgtg
     1081 agtgtggcgc gggagctcgg ccttccggtt gagaattaac tcgagcacca ccaccaccac
     1141 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1201 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1261 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1321 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1381 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1441 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1501 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1561 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1621 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1681 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1741 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1801 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1861 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1921 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1981 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     2041 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     2101 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     2161 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     2221 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     2281 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     2341 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2401 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2461 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2521 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2581 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2641 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2701 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2761 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2821 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2881 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2941 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3001 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3061 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3121 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3181 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3241 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3301 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3361 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3421 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3481 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3541 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3601 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3661 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3721 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3781 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3841 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3901 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3961 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4021 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4081 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4141 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4201 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4261 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4321 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4381 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4441 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4501 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4561 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4621 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4681 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4741 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4801 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4861 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4921 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4981 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     5041 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     5101 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5161 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5221 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5281 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5341 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5401 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5461 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5521 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5581 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5641 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5701 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5761 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5821 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5881 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5941 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     6001 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     6061 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6121 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6181 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     6241 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     6301 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc
     6361 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat g
//
