LOCUS       sequence-227335-        7956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbC-nHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7956)
  AUTHORS   Zha L, Wilson MR, Brotherton CA, Balskus EP
  TITLE     Characterization of Polyketide Synthase Machinery from the pks 
            Island Facilitates Isolation of a Candidate Precolibactin.
  JOURNAL   ACS Chem Biol. 2016 May 20;11(5):1287-95. doi: 
            10.1021/acschembio.6b00014. Epub 2016 Mar 1.
  PUBMED    26890481
REFERENCE   2  (bases 1 to 7956)
  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/acschembio.6b00014"; journalName: "ACS Chem Biol"; date: 
            "2016-05-20- 20"; volume: "11"; issue: "5"; pages: "1287-95"
COMMENT     Sequence Label: pET28a-clbC
FEATURES             Location/Qualifiers
     source          1..7956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..65
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     149..168
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        149..167
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    168..192
                     /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             207..229
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             248..265
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             275..292
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             296..328
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             2969..2986
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3039..3057)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3053..3100
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3137..3592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3224..3243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3434..3455
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3684..4499)
                     /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(3684..4499)
                     /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     4407..4426
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4491..4510
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4621..5209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5110..5129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5363..5380
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5395..5534
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5460..5479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5620..5642
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5636..5827)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6636..7718)
                     /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     7680..7699
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7719..7796)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
       61 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      121 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      181 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      241 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatggc
      301 tagcatgact ggtggacagc aaatgggtcg cggatccgaa ttcgagctcc gtcgacaagc
      361 tttggaatac gcaagcgaaa tgaacggcat ggaaatcgcc attattggta tggcggtccg
      421 tttcccgcag tcccggacgt tacacgaatt ctggcataac atcgttcagg gtaaagagtg
      481 cgtcaccttc tttagcgagg aggagttgct ggccgaaggc gtcgaacaga gtactctgga
      541 caacccggcc tatgtacggg ccaagcccta tatcgagggc atatgcgact ttgatgctgc
      601 atttttcggt tacagtcaca aagaggcgca gactctggat ccgaaatccc gcgtattaca
      661 tgaagttgcc taccatgcgc tggaagatgc aggctatgcc caacgtacca gcgatctgat
      721 caccggggta ttcgtggggg cgtcagaaga tgtggattgg ctacggcgtt cgctgtcaca
      781 gattggcggc gatgcgctga atcgttttga gtctggcatc tatggtcata aggatctgct
      841 cgcacatctc attgcctaca gtctcaatct caacggtccg gtgtatagtc tctacaccag
      901 ttgttcgact tctctgagtg caacgcatat cgcttgccgc agcttgttgt ttggcgaatg
      961 tgatctggcg ttggcgggtg gaattactat cgatttaccg cagaagtcag gctacttctg
     1021 tcaacagggc atgatccact ccaccgatgg ccactgccgt ccttttgaca gtcaggcttc
     1081 tggcaccctg tttggcgatg gcgcgggtgt ggttgtgctt cggcgtttgg aagatgctct
     1141 ggcagcgggc gatcgcatct atgcggtgat ccggggtagc gcggtcaaca atgacggtaa
     1201 acaaaaaatc ggttttgtcg cgcctggtca tgagggacag aaggcggtca tttgtgcggc
     1261 ctgtcatctg gcagaagtaa gcccagaaag catcggctac gttgaaaccc acggtaccgg
     1321 cacccgtatt ggcgatccta tcgagttcgc tgcgttgacg gaggcgttcg atacttcaca
     1381 ccgccaatac tgtgcactag gggctgtaaa ggcgaatatt ggccacaccc acgcggcggc
     1441 gggcgtggct gggttaatca agacggcgct ggtacttcat caccggacca ttccgccgct
     1501 cgccaactat caaatgccta actcgaagct ggatctggcg cattcaccgt tttatatccc
     1561 gatacagccg caggagtggc ccgcatcgcg gatgccgccc cgcgctggcg tcagttcttt
     1621 tggcattggc ggtactaatg tgcatatgat tttggaaggg ctgaatcctg cggtgcgcga
     1681 tgaccatgac caagtgcgag caccggtgtt tatccctctc tccgcgccgt ctttcgagca
     1741 gttggatgag ctgacgcaac agcttacccc gttgctggct acccttgatg cgtcaacgct
     1801 agcctacaca caacaagtgg cgcgccccgt gtttgattgc cgccgagtga tacaagtgga
     1861 aaacgatggt acgcaagcga tgctggcatc gctggataac ctaatgcccg acgctccttg
     1921 gggcctacac tgtccagatc tacgtactac gaacgattgt acttacgcac agtggctggc
     1981 gcattcggca cattatcaac gcgaagcgac tgctctgacg gcattactcg acggcatgaa
     2041 tattccaccc gcttattgcc acgctgaaac gtgggcggca caagcgaaca gcagcctgct
     2101 aatcagaggc tgccagacta tcgccgcgct aaaaacctgg atgaacctgc taccgacatt
     2161 aacactgctg tcgggtgctg gaacaggcct tttgcctgcc gctgcagcca gtggcatgat
     2221 tgcgacgcaa gacgtgttgc atttgctgtg ggaaatggag caaaaggcgc ttcatctctg
     2281 gcttcctgag cgccatgaac ctatccccgg ctacgtgctg gcttggcaag gaaatcccat
     2341 caccgatgca cagcgtaacg acagagggtt ttggagtgaa gcgctgttgg cagatacccg
     2401 agaactgggg gagggcgttc acagtatcaa ctgggttagg ctgccgccgg aaataagaga
     2461 agacgttgat gtattgcgct acgtggcgca actgtggtgt gcaggtatca atgtggattg
     2521 ggccgtgtgg tacggcactc cgctgccgca acgcgggagc gcatcagcat atcccttcgc
     2581 acacaaccac tacccattgc ctggacgggt aatgggtagt gtggagaccc aacccgaagc
     2641 aggacccgaa acgcaccacc cttatcaggc acgtcccgtg ttaagcgtac ctttcgtagc
     2701 cgcacactcg cggggtatgc agtatatcac aggtctgatg gaactgttgc tggaaatatc
     2761 gccggtcggg gtggacgacg acttttttga attaggcggg cattcgctgc tggtgacgca
     2821 attgacctcc cgtttagaac gcgatttcaa cgtacatatc gatcttttga ccctgatgga
     2881 aaaccccaat ccgcgcaata tctacgcgca tatcgcggcg caactggggg gcgaagacaa
     2941 cctcgaaata gcctgtcagt aactcgagca ccaccaccac caccactgag atccggctgc
     3001 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     3061 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     3121 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3181 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3241 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3301 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3361 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3421 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3481 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3541 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     3601 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3661 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     3721 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     3781 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     3841 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     3901 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     3961 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     4021 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     4081 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     4141 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     4201 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     4261 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     4321 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     4381 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     4441 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     4501 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     4561 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4621 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4681 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4741 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4801 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4861 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4921 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4981 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5041 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5101 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5161 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5221 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5281 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5341 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     5401 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     5461 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     5521 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5581 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5641 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     5701 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     5761 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     5821 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5881 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5941 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     6001 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     6061 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     6121 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     6181 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     6241 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     6301 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     6361 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     6421 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     6481 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     6541 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     6601 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6661 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6721 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     6781 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     6841 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     6901 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     6961 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     7021 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     7081 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     7141 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     7201 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     7261 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     7321 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     7381 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     7441 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     7501 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     7561 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     7621 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     7681 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     7741 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     7801 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     7861 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     7921 cccggccacg gggcctgcca ccatacccac gccgaa
//
