LOCUS       sequence-227333-        6101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbD-cHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  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 6101)
  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-clbD
FEATURES             Location/Qualifiers
     source          1..6101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     119..138
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     226..245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        226..244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    245..269
                     /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             284..306
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1187..1204
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1257..1275)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1271..1318
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1355..1810
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1442..1461)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1652..1673
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1902..2717)
                     /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(1902..2717)
                     /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     2625..2644
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2709..2728
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2839..3427
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3328..3347
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3581..3598
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3613..3752
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3678..3697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3838..3860
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3854..4045)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4854..5936)
                     /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     5898..5917
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5937..6014)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
       61 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
      121 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
      181 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
      241 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
      301 aggagatata catatgatga acgtggcggt aataggtgca ggagtaatgg gaactggcgt
      361 cgctcataac atggcgcaat acggcatatc aactaacgtt gtggatattt ctcaatctca
      421 gttggataaa tgccgacaga tgatcgaagc gaacttacgc ttatataatt ttcacccgca
      481 gcataaaaaa aagacgcata gcacagcgga gataatggaa aatatccgtt tcacaactga
      541 gttggacgac attgtagaat gtgatttggt gattgagaat attactgagg atatagagaa
      601 aaaaaatgca ctatacacac ggatgaatac gatctgcggc gcgtcgacag tgtttggtgt
      661 taatacgtcc gccatatcta tcactgcgtt gtcaaaatta atgcgacatc cggagaatgt
      721 agtcggcgtc cattttatga acccggtacc gctgatgcac accgtcgaat taatccgtgg
      781 tgtacatact gcagagcgta cactgaacat atttcaccac ttatttgctc agttgaataa
      841 aaccggcatc gtggtcaatg attctccggg ctttgtcact aatcgtgcca tgatgatttt
      901 tgttaacgaa gccatattta tggtgcagga gcagatcgcc cgtgctgagg atatcgatac
      961 gttgtttaaa acctgcttcg ggcacaagat ggggccacta caaactgccg atttgattgg
     1021 cttggatacg attttgcaat cgttgcaggt gctatatgaa agttttaatg atgataagta
     1081 tcgccccagc tttttattaa agaaaatggt cgatgcaggg tatttgggcg tgaaatcagg
     1141 gcaggggttt tatcgctatc aacagacgta cgccgagcag ctcgagcacc accaccacca
     1201 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1261 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1321 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1381 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1441 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1501 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1561 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1621 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1681 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1741 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1801 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1861 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1921 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1981 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2041 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2101 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2161 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2221 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2281 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2341 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2401 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2461 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2521 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2581 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2641 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2701 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2761 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2821 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2881 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2941 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3001 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3061 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3121 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3181 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3241 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3301 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3361 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3421 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3481 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3541 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3601 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3661 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3721 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3781 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3841 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3901 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3961 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4021 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4081 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4141 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4201 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4261 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4321 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4381 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4441 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4501 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4561 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4621 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4681 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4741 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4801 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4861 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4921 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4981 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5041 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5101 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     5161 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5221 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5281 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5341 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5401 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5461 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5521 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5581 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5641 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5701 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5761 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5821 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5881 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5941 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6001 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6061 gcccagtagt aggttgaggc cgttgagcac cgccgccgca a
//
