LOCUS       sequence-227337-        7691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7691)
  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 7691)
  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-clbO
FEATURES             Location/Qualifiers
     source          1..7691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     127..146
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        127..145
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    146..170
                     /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             185..207
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2678..2695
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2748..2766)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2762..2809
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2846..3301
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2933..2952)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3143..3164
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3393..4208)
                     /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(3393..4208)
                     /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     4116..4135
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4200..4219
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4330..4918
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4819..4838
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5072..5089
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5104..5243
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5169..5188)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5329..5351
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5345..5536)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6345..7427)
                     /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     7389..7408
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7428..7505)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
       61 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
      121 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
      181 taattttgtt taactttaag aaggagatat acatatggca aaggatgatt ttacctgtgg
      241 ctcactggat attgccatta ttggcatgag cgggcgtttt tccggtgcgg aatcggtgcc
      301 cgagtggtgg gataagttgc tggcgggtga ggagttcact caaccgacat gcacagaaga
      361 cgataatggg aacccttgga ttaggctgcg caatataatc accgctccct atgacttcga
      421 tgctgcattt ttcaatattc cgcctggcga agccctgttg atggatccgc aacaacgaat
      481 atttcttgaa tgctgctaca acgctcttga gcatgcgggc tatattccca cgcaacttaa
      541 gcgagtcggc gtctatgggg cgacctacgc caataactat tttatcgatc gcgtctatcc
      601 atacctgaag atgagcggcg atcaccatta tcttcaggcg cagattggta acgaaaagga
      661 ttacttgtgt gctcaggttg cctacaagtt ggggtttacc ggacccgctg tgagtgtaca
      721 gaccgcctgt tccagttcgt tggtggcagc atatttagca tgcgaggggt tattgacttt
      781 tcaagccgat gttgcgttgg caggtggggt caccttgggt tttttacagg cgcacggcta
      841 cagtccgcaa ggtgataagc tggtatcgca agacggacac tgcgccccgt ttagcgctga
      901 ggcgaccggc acggtataca gcagcggtgc gggagttgtg gtgctaaaac gtcttgagga
      961 tgcactacgc gaccaagaca gagtgtatgc ggtgattaaa ggaggtgcgg taaataacga
     1021 cggcgggcgt cggctggggt ttgtcgcccc cagcgtagaa ggacaggtag aagcgatcaa
     1081 tacggcgctt gccgccgcag aagtcgtacc gactgatatc gcactgatcg aaacgcacgg
     1141 taccggtacc ccgttgggtg atgagattga gcttgaagct ctgcaccggg ttttcgcacc
     1201 ggcatgtgcg ccgcacagca ttcaattggg tgcggtaaaa gccaacttgg gtcacctggg
     1261 agtcgcttcc ggtatcgtca gcttaatgaa aaccgcactg acgctatata ctggtttagt
     1321 tcccccgcag atcaacctag taaataaaca taaaaaactg ttgcagccag cctcgccatt
     1381 ttatctcagt gatgtagtga cttctgtccc acaaacgaaa aggatccatg ccaccgttag
     1441 ttcgtttggg ctgggaggga ccaatgctca tttggttctg caaaactggt gcgagacgcc
     1501 tgcgcaagcg gtgcaagaaa atgagcgccg cctgtttttc ttcagcgcca aaacgccatt
     1561 agcccttaga caacagttgg atgctcatta tcacgctctg gcgacctatg cggaagcaga
     1621 taaagatcgt atcgcctata ctctcgcgca acgccgggca catttcccgt atcgctgtgc
     1681 gctggcggca gacagtgtgg tagcgttgcg tgcaagtttg gcgaaactgc gggatgctga
     1741 catgtctttt acccctatca atatggaaac taccttggtg ttcctatatc cggaccggga
     1801 cgataagctg gagagtgccc tgacgcattt gctggcttgt caacctaact tgcgccagcg
     1861 gcaccagcgc ctgtctcagg atgttgcaca aatctgcgaa ccagccgatt ggacgccagc
     1921 attgcgtcag tttatccagc aggtgagtct tagtgaatgg ctgatcgaac aaagtatctc
     1981 gcctgtgcag cacatcggtt acctgacggg ggctgcagca gcgcagtatg ttgcgcgcat
     2041 catctcactg gaaaacgctg ttcagcaggt gattgtggct gaaacgacac cggagcagac
     2101 gctggccggc aacagtgaac tgagcgaaat attggctaat ctggcggtga cggagggaac
     2161 gctgatgctg gaaatcggtc gcgcggggac gttttccata ttgtatcacc agcatgcgca
     2221 gtgggttggg caaacagtat tttcccccat gctgaatacg gatacgcctg aggacatcct
     2281 gccgctgttg ggcactcttt ggcaacgagg agtgactatt tgtttaccgg aaatgccagc
     2341 cgtgcagact ataggcttac ccggttattc gtttgatcgc gtccggtatg aaattcagtc
     2401 cagtgatgcg agagagaacg ctatgttacc agtgagttat ttgtcggtca gcgactttgt
     2461 ggagaaaacc tggcgttcat tgttatgcat cgatcattat gacgaacacg cggttatttt
     2521 cgaatacgga gcgacctcga tgcacgttat ctctttcgtc gacagctgta accacattta
     2581 taaaatcgga ctgactgccg ccgatattta tgccagaccc gctatccgtg agcacagtga
     2641 atttatctcc gaatgtgttg atggtatttt actcgagcac caccaccacc accactgaga
     2701 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     2761 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     2821 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2881 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2941 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3001 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3061 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3121 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     3181 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3241 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     3301 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3361 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     3421 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     3481 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     3541 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     3601 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     3661 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     3721 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     3781 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     3841 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     3901 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     3961 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     4021 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     4081 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     4141 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     4201 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     4261 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4321 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4381 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4441 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4501 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4561 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4621 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4681 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4741 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4801 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4861 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4921 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4981 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5041 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5101 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     5161 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     5221 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     5281 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5341 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     5401 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     5461 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     5521 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     5581 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     5641 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     5701 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     5761 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     5821 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     5881 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     5941 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     6001 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     6061 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     6121 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     6181 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     6241 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     6301 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     6361 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6421 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     6481 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     6541 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     6601 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     6661 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     6721 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     6781 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     6841 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     6901 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     6961 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     7021 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     7081 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     7141 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     7201 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     7261 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     7321 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     7381 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     7441 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     7501 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     7561 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     7621 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     7681 cccgaagtgg c
//
