LOCUS       sequence-219791-        8708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbH-C-A2-PCP S1524A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8708)
  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 8708)
  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-clbH-C-A2-PCP S1524A
FEATURES             Location/Qualifiers
     source          1..8708
                     /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             218..262
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             278..295
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3695..3712
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3765..3783)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3779..3826
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3863..4318
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3950..3969)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4160..4181
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4410..5225)
                     /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(4410..5225)
                     /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     5133..5152
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5217..5236
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5347..5935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5836..5855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6089..6106
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6121..6260
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6186..6205)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6346..6368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6362..6553)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7362..8444)
                     /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     8406..8425
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8445..8522)
                     /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 acatatgaaa gaaaccgctg ctgctaaatt
      241 cgaacgccag cacatggaca gcccagatct gggtaccctg gtgccacgcg gttccatggc
      301 tgatatcgga tccgaattcc tgccgaaccg tttgttggca atgtcagagt taccactctc
      361 cgacaatggc aagtgctgtc gtaaaacgtt gctcgacttg gcgcgggcgt actcagccag
      421 tcgcgtcgat ttacgtgaaa ctcccgccgt gcgctaccta ccgttgtcat cggctcaatc
      481 gtcgatgtgg tttatgcaac aattggcgcc gcatactgca ctatacaata accctaccgc
      541 cttgctgctt gaaggagaac tggatcgcac gcggatggac ggcgcgattc gtcaattgat
      601 gagtcggcat actctgttac gtgctatggc ggaaacccac aatggacaac cagtattggc
      661 ggtgcctcag tgcgtatcgt cgcaggcgtt gctgaccata gtgccactcc cctcggtgag
      721 tgatgataac gcgctacagg cgatgatcaa ccagcgtgcg gcacacccca tgcccttaac
      781 gtcaggcaca ccgctgtgcc ggtttgaact gttgacgctc gatgacgatc gtagcgtatt
      841 gttgattcat ctgcaccaca tcatcagtga tggctggtcg aaaggcgttt tgttacgcga
      901 attgcaggcc gcatataacg gtgaatcgct gacgccagaa ccgttgttgg agtatgccga
      961 ctacatggag taccaagaag agtggcgtca gagtgacgcc tatcaggacg cgatgcgtta
     1021 ctggcaaaat accctggcgg ggacgttgcc gattcttgat atccccaccg atcaaccgcg
     1081 tcagaaggtg gcacgttacc aaggcgcgtt tgttgccttc gcattgtctg ccaacacatg
     1141 cgagcgagtg ttggcggcgg cgcgtgcgca gcgcgtgtcg ttgtataact atctgctgac
     1201 cgccttcgtc ctcctgctgc atcgtaatgc gcgtcaacag gaatacatcg tcggtatgcc
     1261 gattgctgcg cggctgacga aagaacagga gcatatgatc gcgccgctgg tcaacgtact
     1321 accgctgcgc ttacctcttg acgaagcggc atcgttttcc gagttagtac agacgatcag
     1381 aggtattctg tttgccgctt tcaggcacca gcgtctcgaa tttaccgaca tagtgcgcgc
     1441 agtgaatgtg gatcgcagcg ccggacattt cccaatatat cagtgcatgt tccaactcga
     1501 caatatgcct ctggcgagcc cgacactaaa cggtgtcaac gttacaccat tattgttgga
     1561 taccagtgca tctcaggtgg atatttcgct aagtatgcag catatcgatg ggcgcatcac
     1621 cggcactttc gaatacgacg ctgggttata cagtgcggat cgtattcagc acctggtggc
     1681 gcagtggaga gaactgctag atgaggccag cagccaaccc acgcagttag tccgcgattt
     1741 gattcgtttc acccctcgag agcacgcttg gctggcgcgg cacaacgcca ctgaagttgc
     1801 tctgccgccc gtagataacc tgctggcgct agtgttaccg cactgccagc agcgccctac
     1861 acaggtggct ttgcgccacg ctgacgacgc catgacctat ggcgaattgc aacaggccac
     1921 gatgcagatg tgtacttggc tgcgtgcgca aggcgtaaaa cgcggagaat ctgtcgcact
     1981 gcaactgcct ttttgtttcg aattgattat cgcacaattg gcgatcctct ctttgggggc
     2041 gagctatgtg ccgctggatg gtaacgcgcc agcggcccgc aatgccctga tcttggcgca
     2101 ggcaacgccg tgcatgctgc tggtggcgca accgctggaa tcccctcatg ggctgacgat
     2161 accttgggtg ctagtgcctg actggcgtag cctgctgaca gaaataccga acctgccagt
     2221 tagtgttgcg ccagatgctt tagattgcga tgcggtggtg atctttacct ccggtactac
     2281 cgggcagccc aaaggcgtcc gactcagcca acgtaatctg gtcaacttaa ccgcatcctt
     2341 tatatccagc tatcaagtga cgcatcagga tgttctgcta cccattacct cagtcgcgtc
     2401 tgctagcttt gtcggggagg tactgccgct gctcgccgct ggcggtacct tagtattggc
     2461 gcaaaaagcg cagagtttgg atagtgatgc gctcattgcg ctgctggcat ctcagcgggt
     2521 gactatcctg agcaccaccc catcgctttc tgccagcctc tctgtgctgg ctcagtcgat
     2581 gggatcgctg cgcttgtttc tgtgcggtgg tgaagcgtta gagtatgagc agatagcgcc
     2641 gcttctgccg cacatggcag tggttaacgg ttatggcctg accgaaagcg gtatctgctc
     2701 gacctacttt cctgtcgcaa agcgtaggga gcaagaaacg ggagcattgc ccattggccg
     2761 cccgattcag aacacccaag cttatgtggt ggatgcatat aatcgtttgg taccgccagg
     2821 tgcctgtgga gaactctgtt tctctggtct gggcatttcg cccggctacc ttgatgcacg
     2881 acaggatccc gagcgctttg tcgagttgcc ggaataccct ggcgttcggg tgctgaaaac
     2941 tggcgatcgt gctcgttggg caaccgacgg gatgttgttc tatctcggca gacaagatcg
     3001 ccaagtgcag atccgtggat accgagttga actgggcgac atcgaaagcc tactgaaaca
     3061 gcatccggat attgctgatg cgtgggtcga tgtgcgacgc aatgcggcgg caacgccgct
     3121 actagtggcc ttctattgca gcgtcaacgg cgttgcgctg gatgctcagc aattgcgcgt
     3181 atggctcagc ctgcggcttc cgttgcatat gctgccgttg ctctacgtgc cgctgagcgc
     3241 catgccgcta ggtgtaaacg gcaaaatcga tccccagtgc ctgccgctgg tcgatctgcg
     3301 gcagttggaa gggccgggcg agtatgtccc tccggcaacc gaactggaac agcgtctggc
     3361 ggagatctgg cagcagttgc ttggcctgga gcgtgtcggc accaccacaa atttcttcga
     3421 tctcggtgga cacgcacttc tgctagtaca gatgcagcaa tacatcgggc agcagtgcgg
     3481 tcagcacgtg gcgttggttg acctgctgcg cttcactacc atcaaacgct tggcggaatt
     3541 tctgctggcc ccggacgcag cgcaagggac cacaggagat cagacacaac tgcgtgcggc
     3601 gaagcagcgt ttggcctttg gtcacacgcg ctgggcagcc acaaccgaca gtcatcaccg
     3661 gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
     3721 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     3781 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     3841 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3901 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3961 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4021 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     4081 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4141 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4201 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4261 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     4321 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4381 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     4441 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     4501 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     4561 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     4621 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     4681 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     4741 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     4801 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     4861 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     4921 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     4981 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     5041 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     5101 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     5161 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     5221 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     5281 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5341 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5401 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5461 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     5521 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5581 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5641 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5701 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5761 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5821 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5881 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5941 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6001 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     6061 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     6121 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     6181 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     6241 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     6301 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6361 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     6421 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     6481 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     6541 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     6601 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     6661 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     6721 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     6781 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     6841 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     6901 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     6961 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     7021 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     7081 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     7141 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     7201 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     7261 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     7321 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     7381 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     7441 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     7501 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     7561 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     7621 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
     7681 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     7741 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     7801 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     7861 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     7921 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     7981 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     8041 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     8101 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     8161 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     8221 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     8281 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     8341 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     8401 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     8461 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     8521 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     8581 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     8641 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     8701 gaagtggc
//
