LOCUS       sequence-227339-        8300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8300)
  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 8300)
  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-clbI
FEATURES             Location/Qualifiers
     source          1..8300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     162..181
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     269..288
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        269..287
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    288..312
                     /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             327..349
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3429..3446
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3499..3517)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3513..3560
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3597..4052
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3684..3703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3894..3915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4144..4959)
                     /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(4144..4959)
                     /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     4867..4886
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4951..4970
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5081..5669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5570..5589
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5823..5840
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5855..5994
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5920..5939)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6080..6102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6096..6287)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7096..8178)
                     /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     8140..8159
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8179..8256)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
       61 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
      121 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
      181 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
      241 atcgagatcg atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      301 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatga
      361 tggcagagaa tgattttggt atagctatca ttgggatggc ggggcgtttc cctcaagccg
      421 atacggtaca ggcgttttgg gaaaatcttc tggcaagccg agagtgcatt tccttttaca
      481 gcgatgagga actgctggcg atgggaatca gtcctgaatt tgttcagcac ccagattacg
      541 tcaaggccaa aggggaggta gctgacatcg ataaatttga tgctgctttc ttcggtatcg
      601 caccccgaga ggcggagctg atggatcctc agcaccgtgt cctgttggaa accgcgtggg
      661 cagccttcga agatgcgggc tatgtggctg ccgattatcc gggggatgtg ggtatttttg
      721 ctggcaaaag catggattcc tatttgatgc tgaacttgat gccgcacttt aaacgcgttt
      781 tctcttctgg cagtttgcag gcggccattg gtaacgacaa agacagtatc actactacca
      841 tcgcctatca cctcaacctg cgcggcccgg cgatcacggt gcagacttcc tcatcgacat
      901 ccttggtggc ggtgtgcgtg gcatgccaaa gtctgttgac ttggcagtgt gacatggcga
      961 ttgctggcgg agtaacgctg gggccaccag caaaaaccgg ctacttgtcg caggaaggtg
     1021 ggatcaccgc cgccgatggc cactgtcggg cgttttccga taacagcagc ggatttgtgc
     1081 ccggcaccgg cgcggggctg gtggtgttaa agcgtgttga tgaagcgctg cgtgatggcg
     1141 ataacatcta tgcggtcatc aaaggatttg ccgtcaacaa cgacggttcg gagaagatca
     1201 gttataccgc gccgagcgtg gatgcccaag cgcgcgccat tgctcaggca cagcggttgg
     1261 caggtttgac accgcaggat atcacttatg tggaagccca cggtaccggt acgcgtttgg
     1321 gcgatccggt tgagttttct gcgctaagcc aagcctttgc gggcgcgtca caaaagcagt
     1381 actgtgcgct ggggtcggta aaaaccaata tcggccattt ggatacggca gcgggcgtcg
     1441 cagggttgat aaaaaccgcg ctggcagtgc agcagggaat aattcccgca acattgcact
     1501 ttgaacggcc caatgcacag atcgatctga ccaatagtcc gttttacatc aacactacct
     1561 gtcaaccgtg gcaaccggaa agcggcatcc gtcgcgcggg cgtcacctcg cttggcatgg
     1621 gcggcaccaa tgcccatgtt gtgctggaac aagcgccagc cgttgacttg caagcgcgag
     1681 cgcctgtacc tgcctacagt attttgccgt tttctgccaa gaccgacagt gccttatcct
     1741 cggggctggc gcgttttgcc gattttctgc aacatgaatc gctgccggat aggcgggatc
     1801 tggcgtggac actctcacag ggacgtaaag ccttcgcaca tcgcgccgcg ctcgtaacca
     1861 gagatctaca tgctgccggg acgctgttgc agcaggccgc gacagcgccg tttgctcgtg
     1921 gtgtagcgca gacacagctc gggctggggc tgttgttttc cgggcagggt agccaatatc
     1981 agcgcatggg ccatcagctc tatcaggttt ggccggccta cgccgatgcc ttcgaccgtt
     2041 gcgcgacgtt actcgagcgt gaataccaac tggatatccg ccatgagttg ttcagagcag
     2101 aagtctcgtt agcccaaggc gaacgccttg cgcaaacctg cctcacacaa ccgctgctgt
     2161 tcagcgtcga gtatgcctta gcccaattgt ggctcagttg gggaatcacg ccaacggtaa
     2221 tgattggtca ttcgctgggc gaatgggtgg cagcgacgtt ggcaggcgtg ttctctttag
     2281 aggatgcgtt gcgcttggtg gcgcgtcgag cagagctgat gcatcaggca ccaagcggtg
     2341 ccatgttgat ggtggcgctg cccgaagcac agattcgcgc actgattacc gccccgctgg
     2401 cgattgccgc tgttaacgcc cctgactatt cggttatcgc cgggcctacg tcggagatcc
     2461 tcgccgtcag ccagcgtctg acggagcaga acatcatcaa caaacgattg catacctctc
     2521 atgccttcca ctctagcatg atgcaagatg cggcgcaggc gctacgtcag gcatttgaga
     2581 atgtacgact gaacccgccg acgctcacca tcatctctac cgtaaccggc gcgcacgtca
     2641 gtgccgacac tctcaccaca ccggactact ggattgaaca gatgctgatg cctgtgcagt
     2701 tctccgctgc actgcaagag gcgcaagcta catttgatgt cgattttctg gaaatcgggc
     2761 caggtgccac cctgacccag ctgaccaacg gtcatgcatt aggtgatcgt ctcgcgttca
     2821 gctcgctgcc agcaggtgcc cgcagcagcg atgagcacaa acatatctta gataccgtgg
     2881 cagcgctttg ggtgcgaggg cataacatcg atttgtctgc gtttgcaggt gagcaaccgc
     2941 gccgtgtctc tctaccgacc tacgcctttg acaaaatccg ttactgggtc gacagtccag
     3001 aagaacagag gagcgccgta acgccggtgg cggacgcggg aagtgtcatc cctagcgaac
     3061 cgtcggtgcg ccgtcagccg cgtccggcgt tttcggtgcc ttacgcggct ccagagagca
     3121 aaacgcagcg cggcttagtg gcgatttgtg aagcattgct gggaattgac ggcttgggca
     3181 ttgacgacaa cttcttcgaa gctggcggac attcattgat gctgggcatg ttgctggcgc
     3241 aggtacagga acggttcgcc gtcactcttt cctttttcga cgtgatggag gatgccagcg
     3301 tgcgcgcgtt agcgcagttg gtcgagcagg agcagcagga tgatggaggg tccgcacttg
     3361 ccgtgctagt taacgacatg attaatgaga attcgagctc cgtcgacaag cttgcggccg
     3421 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     3481 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     3541 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     3601 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3661 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3721 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3781 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3841 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3901 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3961 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4021 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     4081 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     4141 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     4201 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     4261 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     4321 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     4381 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     4441 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     4501 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     4561 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     4621 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     4681 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     4741 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     4801 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     4861 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     4921 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     4981 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5041 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5101 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5161 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5221 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5281 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5341 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5401 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5461 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5521 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5581 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5641 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5701 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5761 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5821 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     5881 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     5941 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     6001 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6061 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6121 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     6181 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     6241 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     6301 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     6361 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     6421 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     6481 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     6541 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     6601 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     6661 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     6721 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     6781 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     6841 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     6901 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     6961 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     7021 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     7081 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     7141 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     7201 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     7261 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     7321 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     7381 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     7441 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     7501 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     7561 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     7621 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     7681 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     7741 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     7801 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     7861 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     7921 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     7981 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     8041 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     8101 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     8161 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     8221 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     8281 gcgactcctg cattaggaag
//
