LOCUS       sequence-227336-        6362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbF-cHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6362)
  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 6362)
  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-clbF
FEATURES             Location/Qualifiers
     source          1..6362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     104..123
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     211..230
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        211..229
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    230..254
                     /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             269..291
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1433..1450
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1503..1521)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1517..1564
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1601..2056
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1688..1707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1898..1919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2148..2963)
                     /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(2148..2963)
                     /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     2871..2890
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2955..2974
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3085..3673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3574..3593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3827..3844
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3859..3998
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3924..3943)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4084..4106
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4100..4291)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5100..6182)
                     /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     6144..6163
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6183..6260)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
       61 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
      121 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      181 ggatcgagat cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
      241 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat
      301 gtgcactgaa aattatgagc tggctcagca agaggcagtc ctgtttgcca aacaacatct
      361 cgccctggct gcacagaata ttgaacgtca gcagtttatt gtgccagaca ttatttcttg
      421 cgtggcgcag gccgggtact taggtgcgtc aatcccccaa aaatatggcg gacgaggtta
      481 cgattcttat caactgtgcg ctttgcatga agtgatggct ggggttcacg gttcgttaga
      541 aaatctcata acagtgactg gcatggtcag cacgctgctg caacgcgtgg gcagcgcagc
      601 gcaaaaagcc cattatttgc caaagctggc aacgggggaa ttgatcggcg cgattgccct
      661 cacagagccg aatatcggca gtgacttagt gaacgtggaa acagaattac agcaggatgg
      721 cgacggttgg cggctgaacg ggaaaaagaa atggatcacg ctagggcaga ttgctgattt
      781 ttttattgtt ttaatccact gtggtaatca attagcgaca gtgcttatcg atcgcaatac
      841 cgacggcttt actatcactc cactcaacga catgttgggg ttacgcggca atatgttggc
      901 agagctgcat tttaatgact gccgcctgaa ggaggatgca ttgctcggtc cgctcacccc
      961 tggggtaccg ctggcggtga attttgcgct caatgaaggg cggttcacca ccgcttgtgg
     1021 cagcctagga ttatgtcggg ccgctgtgga tgtggcagcg cgctacatcc gccagcgtaa
     1081 acagttcaag cggcggttat tttctcatgg cattgttcag cacttgtttg ccacaatgct
     1141 gacccaaacg cgcagtgcgc aactaatgtg cttcagcgcg gcggaatacc gcgaaacgct
     1201 gcacccggcc atgataaacc aaatcctgat ggcgaagtat gtcgcttcca aagctgcggt
     1261 ggacgtggcc gggaaggcgg tgcaactgct cggtgcgaat ggttgccatg ccgattatgc
     1321 cgtcgaacgt tactaccgcg acgccaaaat catggaaatc atcgaaggga catcgcaaat
     1381 tcatgaaatc caaattgcga tgaattacat gatggggagt gaagcactcg agcaccacca
     1441 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     1501 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1561 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     1621 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1681 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     1741 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     1801 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     1861 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     1921 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     1981 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2041 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2101 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     2161 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     2221 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     2281 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     2341 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     2401 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     2461 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     2521 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     2581 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     2641 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     2701 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     2761 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     2821 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     2881 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     2941 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     3001 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3061 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3121 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3181 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3241 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3301 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3361 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3421 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3481 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3541 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3601 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3661 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3721 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3781 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3841 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3901 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3961 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4021 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4081 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4141 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4201 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4261 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4321 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     4381 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4441 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4501 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4561 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4621 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4681 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4741 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4801 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4861 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4921 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4981 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5041 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5101 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5161 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5221 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     5281 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5341 catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
     5401 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5461 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5521 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5581 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5641 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5701 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5761 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5821 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5881 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5941 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6001 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6061 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6121 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6181 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6241 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6301 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6361 ca
//
