LOCUS       sequence-72776-e        8601 bp    DNA     circular SYN 14-APR-2025
DEFINITION  NRPS module of ClbB containing CAT domains with N-terminal 6His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8601)
  AUTHORS   Brotherton CA, Balskus EP
  TITLE     A prodrug resistance mechanism is involved in colibactin 
            biosynthesis and cytotoxicity.
  JOURNAL   J Am Chem Soc. 2013 Mar 6;135(9):3359-62. doi: 10.1021/ja312154m. 
            Epub 2013 Feb 20.
  PUBMED    23406518
REFERENCE   2  (bases 1 to 8601)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1021/ja312154m";
            journalName: "J Am Chem Soc"; date: "2013-03-6- 6"; volume: "135"; 
            issue: "9"; pages: "3359-62"
COMMENT     Sequence Label: pet28a-ClbB-NRPS-NHis
FEATURES             Location/Qualifiers
     source          1..8601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(559..1374)
                     /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(559..1374)
                     /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     1282..1301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1366..1385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1496..2084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1985..2004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2238..2255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2270..2412
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2338..2357)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2498..2520
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2514..2705)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3514..4596)
                     /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     4558..4577
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4597..4674)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4880..4899
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4983..5002
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4983..5001
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5002..5026
                     /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             5041..5063
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5082..5099
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5109..5126
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5130..5162
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             8445..8462
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8515..8533)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8529..8576
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2281 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     2341 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     2401 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     2461 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     2521 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     2581 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     2641 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     2701 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     2761 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     2821 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     2881 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     2941 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3001 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3061 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     3121 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     3181 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     3241 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     3301 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     3361 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     3421 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3481 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3541 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3601 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3661 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3721 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3781 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3841 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3901 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3961 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4021 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4081 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4141 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4201 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4261 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4321 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4381 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4441 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4501 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4561 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4621 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     4681 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     4741 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     4801 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     4861 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     4921 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     4981 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     5041 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
     5101 gcagcggcct ggtgccgcgc ggcagccata tggctagcat gactggtgga cagcaaatgg
     5161 gtcgcggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcatggat aatacctctg
     5221 gagattttcc atgtaataag atggacacgc gtaagcagtt accgctaaca ccaagtcaac
     5281 aggggttttt attccattcc ttaaaggata agaaaaggag taactaccat gagcatttta
     5341 catgcatttt ttctcagcat gtagatagcg cccacttcaa gtgggcgctg gaaacgttat
     5401 ttcgaaagca tgagtgtttt cgcactgatt ataactggga gattgatgag cgcccttgtc
     5461 aggtggtgaa gaccgatgtg ttgccggata tatatgtgtt agactgtgag caagaggaaa
     5521 tacgttttct actagctaat gatgacatta tcattcctgt cccgcaggat gacggtattg
     5581 atgctataat tcctcaactg ctacaggctg atttaaaata cccattttcc ttgaaaacga
     5641 tcccagtccg ggcctacctt attcagtcaa cgaaagaaag tgcttttata ctatcatacc
     5701 atcatattgt gatggatggc tggagcttat cccttttcat taaacagttg ctccaactct
     5761 atggagcggc tgtggtcagt ggtgtgaggg atgatagcgc cattatcccc tcatctctga
     5821 aaccccttgt agacacactg tcggcccgac gtcacacctt tcagcacgac tattgggctg
     5881 catatcttcg ggagggaaca ccaacttgta tcgtgccgct gtcacaatat cacacagata
     5941 ctgaagccga gaacaattct tacgttaatc aaacaaatca tgtggagatc aatttgtctc
     6001 cggatgtgtg tcagaaaata cagacgctat gcagcgatta tcgtatcacc cccgcagtaa
     6061 tcttctatgt ggcctggggc atcctgctac aacgttggtg ctatgctgac gatgtgttat
     6121 tcggcgcgac aatatcaggg cgaaatatac caattgatgg tatagaagaa acactagggc
     6181 tatttattaa cacgttgccg ctgcgtctgc gtgatgatgg ggcgacactg ttgcaacact
     6241 tacaacgtat gcaccaaaca ctaatagctc actacagcaa tgaacatgat gccttagcca
     6301 gcatacaacg gttggtacat aaagaaggtc atgctgggga tctttttaat accttagtgg
     6361 tgttggagaa ttatcccgtt gatatgacat tattgtcatg cgcgtcgccc gtggcaattc
     6421 gccatctcag tgtacatgaa caaacgcact atccgctgac cttgaccatc actcaacaga
     6481 agggattccg tttcagtatt gcatatgccc ttaactacct gaccaacaac atggcgcaag
     6541 cgttgctgat gcacctgagt tatctgcttg aacaactggt ggacaatccg cagcgcccca
     6601 ttgctgcgtt ggtaaacttg tccccatgcc agcaggcgca ggtacttcaa ccctatctgg
     6661 aacgcatggc atgccgggat tgggatagtc aatccaacgt catcgaacaa tttcatcaag
     6721 ttgctgcgac ttcgccagca caggtcgcag tggttgatga attgtgcgcg ttgacctatt
     6781 cggagttggc agcacaagct gagcagctcg cggcttattt ggtacagcag ggtgttatgg
     6841 ttggcgatac tgtaggcata attagcgaac gccgggtaaa caccgtggtt gccatcatcg
     6901 ccatcatgtt gatcggggct gcttatgtgc ccatcagccc cgactaccca gtgggtagga
     6961 tgcaggaaat tattgatgac agcggcttgg cgctgttgct ggtacatggc aaaccgctag
     7021 atgcattaaa cgttgcgcag agtgacctct gtgcatttcc cgtcgcgccc tcggtggtat
     7081 ttccggttat cacaccagat tctcgcgctt atgtgattta ttcatcgggg tcgacgggta
     7141 agccaaaagg tatcgcggtg gcgcaccgcg gtttattgcg cctgatacaa ggcgacagcc
     7201 cgctgaaggt ggagagcggt gagacaacgc tgctgacctg cccatttgag tttgatgttt
     7261 cggtgtttga gatgtggtct accttgctca accacggcaa actagtatta ctcagcaaac
     7321 aggcattact tgatatcaat cacattcgcc gcacgatcgc tgatgaacag gtggcgcgcg
     7381 cctggtttac ctcatccttg tttaactcct atgtggcaga aggtgccgat ttcttcggta
     7441 tgttacaaca cattacggtg ggcggcgaag cagtatctgc gtggcatgtc aacgacgtga
     7501 tgcaaaaata cccgcatctg gtggtgacaa acggttatgg gccgacggaa aacactattt
     7561 ttaccaccgc atatcgtttc aacgggttgc aacccgcccg agtcccgata ggatacgcgg
     7621 taccgggcac ctcgctctac attaccgatc tccatgggca tttgttgcct atcggtgcca
     7681 ccggtgaact agtggcgggt ggagtggggg tcgccatcgg ctatcagaac aacccggcgc
     7741 taagtgcgac ggtttttgtc cctgatcctt ttattcccgg cggtatgatg tacaaaactg
     7801 gcgattatgc ccggttgctg gatgatggct gtgttgactg ttttgggcgt aaagacggtc
     7861 aaattaagat caatggacaa cgaattgaaa ccggagagat cgagcagcgc ctgctggagt
     7921 gctccggcat tatcgaggcg gtagtggttc cttaccgcgt acgtgaaacg ctgcatattg
     7981 cggcagtggt ctgcgtcaat gatagctatg atgaggtgga agttcgaggg caattggctg
     8041 acagattgcc gccattcgct attccggaat ccttggtggt ggtgacggag attgcaaaaa
     8101 gccatagtgg caaggccgac ttggcgcagt tgcggtatct cctgcccgca actcagtgca
     8161 acgccgtgtc caccacgata tcagaggtgc atagtgacat ggaacatgcg ctgcatgcta
     8221 tctggcaacg cgtacttgat agacaagaca ttgatagcaa tgcctccttt ttcgccctcg
     8281 gtggcacctc attggatacc atcagggtta aaggggatat taagcggcaa cttggcttgg
     8341 agattgatat taccgatctc tttaagtacc caacgctcac ggcgttagca cattttctcg
     8401 atactgccgt atcgccggag gatgcaattc caacgtgact cgagcaccac caccaccacc
     8461 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     8521 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     8581 aaggaggaac tatatccgga t
//
