LOCUS       sequence-227348-        6940 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ClbH-A1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6940)
  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 6940)
  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: pET28a-clbH-A1
FEATURES             Location/Qualifiers
     source          1..6940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     203..222
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        203..221
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    222..246
                     /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             261..283
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             302..319
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             329..346
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             350..382
                     /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             2007..2024
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2077..2095)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2091..2138
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2175..2630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2262..2281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2472..2493
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2722..3537)
                     /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(2722..3537)
                     /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     3445..3464
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3529..3548
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3659..4247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4148..4167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4401..4418
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4433..4572
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4498..4517)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4658..4680
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4674..4865)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5674..6756)
                     /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     6718..6737
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6757..6834)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
       61 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
      121 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
      181 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
      241 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag
      301 ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata tggctagcat
      361 gactggtgga cagcaaatgg gtcgcggatc cgaattcatg gaacagcaag ggattatgag
      421 acagttgcct accgacgacc aaacgatagt cgactatttg tatcgtatcg ccggagaata
      481 tggggaaaaa gccgctgtat tgatggggga cgcggcgctg agctatcacg atcttaatgc
      541 acgctctaac caactggcgc attatctgcg tgggctgggg atcggcgagg atcgtgtggt
      601 agctatccgc ctgccgcgcg gcatggcaat gctgattgcc attttcgcta ttgtaaaagc
      661 tggtggtgcc tatttaccgc tggcgtacaa tgcaccgcgt agccgcattg aaaatatact
      721 gagcaacagc ggcgctgttt gtctgatcgg tactgacgat ggtgatcgct ggccgattcc
      781 tcgcgtcgaa atcgacagcg cggcggtcag tgccatgcca acgacggatc tgcgttaccg
      841 accacatgcg cggcaactgg cgtatattat ttacacctcc ggttccaccg gtgtccctaa
      901 aggggtggcg acggaacatg cagcgctgct aaaccgaatt gtctggatgc agaacgctta
      961 tcctatcagt tctcaggacg tgctgtttca gaagacggtg tacacctttg acgtctctgt
     1021 ctgggagatg ttctggtggg cgatgtacgg cgcatctgta gtgctgttac cgtccggact
     1081 ggagagcgat ccgcgaacct tggctcgact gattcagcgt caccgcgtgt cggttgtgca
     1141 ctttgtccct tcgatgctga acctgtttgt cgagtatctg gagatgaaac aggatcctcg
     1201 tttgaccgcc tcattgcgat tagtgttttc cagcggtgag aaactcacgg tccacagtgt
     1261 ggctcgcttt tatcaatcgg tggcgcaggg tgatcttata aatctctatg gcccgaccga
     1321 agcagcaatc gatgtcagtc atcaccgctg cctgcgcggg tacgactacg acgatatccc
     1381 tatcggtcaa gcgattgacg gttgccgact ctatgtgctg gatgaccatg gtaatccggt
     1441 agcagacggc gaagagggcg aactgtatct cgctggtatc gggctggcac gtggctatct
     1501 caacaacgtg gcgttaactg atcgctgttt tactatacat ccaactttgc gccatttagg
     1561 gaaaccggag cggctgtata aaactggaga tctggtgtgg cgcgacgggg aaagccaaca
     1621 aattcattac attggccgta atgattttca gattaaaatt agagggttgc gcgttgaatt
     1681 gggagaaatc gaagcccatg cgatgcgttt cccgggggta cagcaggcag tcgtggtggc
     1741 ggatcaggat gatcccgaca atcagttgat ttacgctttt gtcgtcagta gcgtgccgct
     1801 caatttggcg gccttaatgg acgcactgtc caaaaacttg cctgcctaca tgctgccgaa
     1861 ccgtttgttg gcaatgtcag agttaccact ctccgacaat ggcaagtgct gtcgtaaaac
     1921 gttgctcgac ttggcgcggg cgtactcagc cagtcgcgtc gatttacgtt aggagctccg
     1981 tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     2041 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2101 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2161 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2221 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2281 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2341 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2401 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2461 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2521 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2581 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     2641 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2701 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2761 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2821 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2881 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2941 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     3001 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3061 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     3121 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3181 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3241 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3301 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3361 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3421 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3481 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     3541 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     3601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3781 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4141 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4201 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4261 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4321 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4381 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4441 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4501 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4561 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4621 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4681 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     4741 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     4801 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     4861 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4921 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4981 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     5041 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5101 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5161 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5221 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5281 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     5341 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     5401 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5461 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5521 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     5581 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5641 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5701 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5761 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5821 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5881 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5941 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     6001 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     6061 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     6121 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     6181 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     6241 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     6301 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     6361 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     6421 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     6481 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     6541 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     6601 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     6661 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     6721 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     6781 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     6841 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     6901 cgttgagcac cgccgccgca aggaatggtg catgcaagga
//
