LOCUS       sequence-227331-        6500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6500)
  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 6500)
  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-clbG
FEATURES             Location/Qualifiers
     source          1..6500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     56..75
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     163..182
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        163..181
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    182..206
                     /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             221..243
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1523..1540
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1593..1611)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1607..1654
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1691..2146
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1778..1797)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1988..2009
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2238..3053)
                     /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(2238..3053)
                     /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     2961..2980
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3045..3064
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3175..3763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3664..3683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3917..3934
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3949..4088
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4014..4033)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4174..4196
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4190..4381)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5190..6272)
                     /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     6234..6253
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6273..6350)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
       61 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
      121 gtccggcgta gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag
      181 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
      241 agatatacat atgacgaagg atgtcgcact gatgttccct ggctccggtt cgcaatatgt
      301 aggcatggca cggtggctgt atgagcgtta tccgcaggtg cgtactctgt ttgatgaagc
      361 tagccagatc acggaacggg acatggcagc gctgtgcctg tcgggtactt tggtacaact
      421 tgctgagccg acggcaatgg cgctggctat ctataccacc agcgtggccc actttgtcgc
      481 gtggcagcag tttttggcgc aaaccggtgt ccatgtcaac ctacgttata tgttgggtca
      541 tagtttgggc gaatatgctg cgctgacctg tagtggtgcg ctgagctttt cccaagcact
      601 ggcgctggtg gcgatgcgta gccgtttagc cagtgagatc gcccgcgaaa tggacgcgag
      661 taccactatc atcaagcaag ggaatcaggc actggtggca gcggcgtgcg aggtggcgga
      721 gcgtgaaacc cgacagcagg tcggtattgc ctgcttcaat tcgccgcaac agtttatgtt
      781 atccggacaa aactcggcca ttatcgccgc cgagcaatac ctgctggatc atgatcgcca
      841 agtcgaagta gtgccgctga ttggtggtgt tccttaccac agcccactat tgaaaccttg
      901 cggacagcaa ttgcgcaagg cgctggatcg ctgcgaatgg cgacgtccgt gctgcccagt
      961 gatctccaat gtcaacgctc aaccctatcc cgatacggtc acgccagtcc agtggctaga
     1021 acaacagctc tctcagccag tgcagtggca gcgctctctc acctacctga caggacactt
     1081 gtcaccgatc gcgatagaga tcggtccgca aagtgtgctg aaaaacctgc tgctggagaa
     1141 tcgctatcca gcaccagttt acgcatttga caatcgtcat gatcgtgcgc aactagcatt
     1201 ggtgctgggg gataacatgg cggtgaagac cgatccggaa gccgtgcgcc gccaacgcat
     1261 cacgctgctc actaacgccc tgacagccac acgccatcac cgcgccgccg atgtggctgc
     1321 cagtgcgcaa ttgaaagaat tgctcagtcg tttttttgag cgcatccagc agattgagca
     1381 gcgcggcaca tccagcgaag aggacattgc ttttttgcat gagcttcttg aacagggatt
     1441 tcaactcaaa ggcagtagtc aggcagaaat cgacgcctgc cacgcgcggt tggcttccga
     1501 caacggcgga caggcgctcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     1561 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     1621 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     1681 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1741 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1801 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1861 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1921 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1981 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2041 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2101 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     2161 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2221 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2281 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     2341 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     2401 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     2461 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     2521 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2581 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     2641 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     2701 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     2761 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     2821 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     2881 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     2941 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     3001 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     3061 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     3121 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3181 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3241 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3301 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3361 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3421 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3481 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3541 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3601 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3661 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3721 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3781 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3841 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3901 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3961 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     4021 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     4081 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4141 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4201 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     4261 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4321 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4381 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4441 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4501 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4561 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4621 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4681 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4741 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4801 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4861 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4921 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4981 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5041 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     5101 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5161 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5221 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5281 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5341 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5401 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5461 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5521 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5581 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5641 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5701 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5761 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5821 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5881 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5941 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     6001 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     6061 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     6121 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     6181 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6241 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6301 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6361 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6421 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6481 cacggggcct gccaccatac
//
