LOCUS       sequence-220643-        8355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbI S178A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8355)
  AUTHORS   Zha L, Jiang Y, Henke MT, Wilson MR, Wang JX, Kelleher NL, Balskus 
            EP
  TITLE     Colibactin assembly line enzymes use S-adenosylmethionine to build a
            cyclopropane ring.
  JOURNAL   Nat Chem Biol. 2017 Oct;13(10):1063-1065. doi: 
            10.1038/nchembio.2448. Epub 2017 Aug 7.
  PUBMED    28805802
REFERENCE   2  (bases 1 to 8355)
  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: 
            "10.1038/nchembio.2448"; journalName: "Nat Chem Biol"; date: 
            "2017-10"; volume: "13"; issue: "10"; pages: "1063-1065"
COMMENT     Sequence Label: pET29b-clbI S178A
FEATURES             Location/Qualifiers
     source          1..8355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        42..60
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    61..85
                     /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             100..122
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             141..158
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             168..185
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3261..3278
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3331..3349)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3345..3392
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3429..3884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3516..3535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3726..3747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3976..4791)
                     /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(3976..4791)
                     /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     4699..4718
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4783..4802
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4913..5501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5402..5421
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5655..5672
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5687..5826
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5752..5771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5912..5934
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5928..6119)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6928..8010)
                     /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     7972..7991
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8011..8088)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8294..8313
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
       61 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
      121 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
      181 gcagccatat gatggcagag aatgattttg gtatagctat cattgggatg gcggggcgtt
      241 tccctcaagc cgatacggta caggcgtttt gggaaaatct tctggcaagc cgagagtgca
      301 tttcctttta cagcgatgag gaactgctgg cgatgggaat cagtcctgaa tttgttcagc
      361 acccagatta cgtcaaggcc aaaggggagg tagctgacat cgataaattt gatgctgctt
      421 tcttcggtat cgcaccccga gaggcggagc tgatggatcc tcagcaccgt gtcctgttgg
      481 aaaccgcgtg ggcagccttc gaagatgcgg gctatgtggc tgccgattat ccgggggatg
      541 tgggtatttt tgctggcaaa agcatggatt cctatttgat gctgaacttg atgccgcact
      601 ttaaacgcgt tttctcttct ggcagtttgc aggcggccat tggtaacgac aaagacagta
      661 tcactactac catcgcctat cacctcaacc tgcgcggccc ggcgatcacg gtgcagactt
      721 ccgcatcgac atccttggtg gcggtgtgcg tggcatgcca aagtctgttg acttggcagt
      781 gtgacatggc gattgctggc ggagtaacgc tggggccacc agcaaaaacc ggctacttgt
      841 cgcaggaagg tgggatcacc gccgccgatg gccactgtcg ggcgttttcc gataacagca
      901 gcggatttgt gcccggcacc ggcgcggggc tggtggtgtt aaagcgtgtt gatgaagcgc
      961 tgcgtgatgg cgataacatc tatgcggtca tcaaaggatt tgccgtcaac aacgacggtt
     1021 cggagaagat cagttatacc gcgccgagcg tggatgccca agcgcgcgcc attgctcagg
     1081 cacagcggtt ggcaggtttg acaccgcagg atatcactta tgtggaagcc cacggtaccg
     1141 gtacgcgttt gggcgatccg gttgagtttt ctgcgctaag ccaagccttt gcgggcgcgt
     1201 cacaaaagca gtactgtgcg ctggggtcgg taaaaaccaa tatcggccat ttggatacgg
     1261 cagcgggcgt cgcagggttg ataaaaaccg cgctggcagt gcagcaggga ataattcccg
     1321 caacattgca ctttgaacgg cccaatgcac agatcgatct gaccaatagt ccgttttaca
     1381 tcaacactac ctgtcaaccg tggcaaccgg aaagcggcat ccgtcgcgcg ggcgtcacct
     1441 cgcttggcat gggcggcacc aatgcccatg ttgtgctgga acaagcgcca gccgttgact
     1501 tgcaagcgcg agcgcctgta cctgcctaca gtattttgcc gttttctgcc aagaccgaca
     1561 gtgccttatc ctcggggctg gcgcgttttg ccgattttct gcaacatgaa tcgctgccgg
     1621 ataggcggga tctggcgtgg acactctcac agggacgtaa agccttcgca catcgcgccg
     1681 cgctcgtaac cagagatcta catgctgccg ggacgctgtt gcagcaggcc gcgacagcgc
     1741 cgtttgctcg tggtgtagcg cagacacagc tcgggctggg gctgttgttt tccgggcagg
     1801 gtagccaata tcagcgcatg ggccatcagc tctatcaggt ttggccggcc tacgccgatg
     1861 ccttcgaccg ttgcgcgacg ttactcgagc gtgaatacca actggatatc cgccatgagt
     1921 tgttcagagc agaagtctcg ttagcccaag gcgaacgcct tgcgcaaacc tgcctcacac
     1981 aaccgctgct gttcagcgtc gagtatgcct tagcccaatt gtggctcagt tggggaatca
     2041 cgccaacggt aatgattggt cattcgctgg gcgaatgggt ggcagcgacg ttggcaggcg
     2101 tgttctcttt agaggatgcg ttgcgcttgg tggcgcgtcg agcagagctg atgcatcagg
     2161 caccaagcgg tgccatgttg atggtggcgc tgcccgaagc acagattcgc gcactgatta
     2221 ccgccccgct ggcgattgcc gctgttaacg cccctgacta ttcggttatc gccgggccta
     2281 cgtcggagat cctcgccgtc agccagcgtc tgacggagca gaacatcatc aacaaacgat
     2341 tgcatacctc tcatgccttc cactctagca tgatgcaaga tgcggcgcag gcgctacgtc
     2401 aggcatttga gaatgtacga ctgaacccgc cgacgctcac catcatctct accgtaaccg
     2461 gcgcgcacgt cagtgccgac actctcacca caccggacta ctggattgaa cagatgctga
     2521 tgcctgtgca gttctccgct gcactgcaag aggcgcaagc tacatttgat gtcgattttc
     2581 tggaaatcgg gccaggtgcc accctgaccc agctgaccaa cggtcatgca ttaggtgatc
     2641 gtctcgcgtt cagctcgctg ccagcaggtg cccgcagcag cgatgagcac aaacatatct
     2701 tagataccgt ggcagcgctt tgggtgcgag ggcataacat cgatttgtct gcgtttgcag
     2761 gtgagcaacc gcgccgtgtc tctctaccga cctacgcctt tgacaaaatc cgttactggg
     2821 tcgacagtcc agaagaacag aggagcgccg taacgccggt ggcggacgcg ggaagtgtca
     2881 tccctagcga accgtcggtg cgccgtcagc cgcgtccggc gttttcggtg ccttacgcgg
     2941 ctccagagag caaaacgcag cgcggcttag tggcgatttg tgaagcattg ctgggaattg
     3001 acggcttggg cattgacgac aacttcttcg aagctggcgg acattcattg atgctgggca
     3061 tgttgctggc gcaggtacag gaacggttcg ccgtcactct ttcctttttc gacgtgatgg
     3121 aggatgccag cgtgcgcgcg ttagcgcagt tggtcgagca ggagcagcag gatgatggag
     3181 ggtccgcact tgccgtgcta gttaacgaca tgattaatga gaattcgagc tccgtcgaca
     3241 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     3301 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     3361 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     3421 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3481 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3541 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3601 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3661 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3721 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3781 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3841 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     3901 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3961 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     4021 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     4081 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     4141 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     4201 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     4261 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     4321 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     4381 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     4441 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     4501 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     4561 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     4621 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     4681 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     4741 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     4801 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     4861 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4921 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4981 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5041 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5101 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5161 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5221 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5281 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5341 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5401 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5461 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5521 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5581 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5641 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     5701 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     5761 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     5821 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5881 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5941 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     6001 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     6061 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     6121 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     6181 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     6241 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     6301 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     6361 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     6421 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     6481 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     6541 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     6601 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     6661 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     6721 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     6781 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     6841 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     6901 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6961 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     7021 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     7081 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     7141 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     7201 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     7261 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     7321 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     7381 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     7441 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     7501 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     7561 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     7621 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     7681 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     7741 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     7801 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     7861 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     7921 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     7981 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     8041 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     8101 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     8161 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     8221 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     8281 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     8341 tgatgccggc cacga
//
