LOCUS       sequence-227328-        5480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbE-cHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5480)
  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 5480)
  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: pET29b-clbE
FEATURES             Location/Qualifiers
     source          1..5480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     62..81
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     169..188
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        169..187
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    188..212
                     /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             227..249
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             509..526
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(579..597)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      593..640
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      677..1132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(764..783)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     974..995
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1224..2039)
                     /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(1224..2039)
                     /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     1947..1966
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2031..2050
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2161..2749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2650..2669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2903..2920
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2935..3074
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3000..3019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3160..3182
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3176..3367)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4176..5258)
                     /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     5220..5239
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5259..5336)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
       61 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
      121 cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta atacgactca
      181 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
      241 agaaggagat atacatatga aaaagcaaga tatgaaagcc gccattcggg aatttctttc
      301 acgctcatta cgtgggcata cgttgaacga tgatgacgat attttttctc tcgggcttgt
      361 ccattcgtta tttactgtgc aaatcatact gtttatagaa aaaaattttc aggttgagct
      421 ggaagtgagt gagttgaaaa cagaacagat tgctaccgtc aataaaatag tggagctcat
      481 tcagcgacaa acaggcctgg agctcgagca ccaccaccac caccactgag atccggctgc
      541 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
      601 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
      661 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
      721 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
      781 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
      841 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
      901 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
      961 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1021 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1081 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     1141 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1201 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     1261 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1321 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     1381 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     1441 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     1501 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     1561 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     1621 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     1681 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     1741 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     1801 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     1861 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     1921 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     1981 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     2041 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     2101 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2161 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2221 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2281 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2341 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2401 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2461 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2521 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2581 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2641 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2701 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2761 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2821 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2881 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     2941 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3001 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3061 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3121 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3181 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3241 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3301 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3361 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3421 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3481 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3541 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     3601 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     3661 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     3721 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     3781 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     3841 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     3901 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     3961 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4021 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4081 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4141 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4201 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4261 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4321 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4381 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4441 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4501 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4561 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4621 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4681 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4741 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4801 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4861 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4921 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4981 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5041 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5101 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5161 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5221 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5281 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5341 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5401 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5461 cccggccacg gggcctgcca
//
