LOCUS       sequence-209047-        8662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PdR-PCNA3(R112C/T180C)-P450cam fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8662)
  AUTHORS   Haga T, Hirakawa H, Nagamune T
  TITLE     Artificial Self-Sufficient Cytochrome P450 Containing Multiple 
            Auxiliary Proteins Demonstrates Improved Monooxygenase Activity.
  JOURNAL   Biotechnol J. 2018 Dec;13(12):e1800088. doi: 10.1002/biot.201800088.
            Epub 2018 Aug 14.
  PUBMED    30039932
REFERENCE   2  (bases 1 to 8662)
  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"; journalName: "Biotechnol 
            J."; date: "2018-12"; volume: "13(12):e1800088. doi"; pages: " 
            10.1002/biot.201800088. Epub 2018 Aug 14"
COMMENT     Sequence Label: pET28b+R3(R112C/T180C)C
FEATURES             Location/Qualifiers
     source          1..8662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3527..3544
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3597..3615)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3611..3658
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3695..4150
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3782..3801)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3992..4013
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4242..5057)
                     /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(4242..5057)
                     /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     4965..4984
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5049..5068
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5179..5767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5668..5687
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5921..5938
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5953..6092
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6018..6037)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6178..6200
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6194..6385)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7194..8276)
                     /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     8238..8257
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8277..8354)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8560..8579
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg aacgcaaacg acaacgtggt catcgtcggt
      181 accggactgg ctggcgttga ggtcgccttc ggcctgcgcg ccagcggctg ggaaggcaat
      241 atccggttgg tgggggatgc gacggtaatt ccccatcacc taccaccgct atccaaagct
      301 tacttggccg gcaaagccac agcggaaagc ctgtacctga gaaccccaga tgcctatgca
      361 gcgcagaaca tccaactact cggaggcaca caggtaacgg ctatcaaccg cgaccgacag
      421 caagtaatcc tatcggatgg ccgggcactg gattacgacc ggctggtatt ggctaccgga
      481 gggcgtccaa gacccctacc ggtggccagt ggcgcagttg gaaaggcgaa caactttcga
      541 tacctgcgca cactggagga cgccgagtgc attcgccggc agctgattgc ggataaccgt
      601 ctggtggtga ttggtggcgg ctacattggc cttgaagtgg ctgccaccgc catcaaggcg
      661 aacatgcacg tcaccctgct tgatacggca gcccgggttc tggagcgggt taccgccccg
      721 ccggtatcgg ccttttacga gcacctacac cgcgaagccg gcgttgacat acgaaccggc
      781 acgcaggtgt gcgggttcga gatgtcgacc gaccaacaga aggttaccgc cgtcctctgc
      841 gaggacggca caaggctgcc agcggatctg gtaatcgccg ggattggcct gataccaaac
      901 tgcgagttgg ccagtgcggc cggcctgcag gttgataacg gcatcgtgat caacgaacac
      961 atgcagacct ctgatccctt gatcatggcc gtcggcgact gtgcccgatt tcacagtcag
     1021 ctctatgacc gctgggtgcg tatcgaatcg gtgcccaatg ccttggagca ggcacgaaag
     1081 atcgccgcca tcctctgtgg caaggtgcca cgcgatgagg cggcgccctg gttctggtcc
     1141 gatcagtatg agatcggatt gaagatggtc ggactgtccg aagggtacga ccggatcatt
     1201 gtccgcggct ctttggcgca acccgacttc agcgttttct acctgcaggg agaccgggta
     1261 ttggcggtcg atacagtgaa ccgtccagtg gagttcaacc agtcaaaaca aataatcacg
     1321 gatcgtttgc cggttgaacc aaacctactc ggtgacgaaa gcgtgccgtt aaaggaaatc
     1381 atcgccgccg ccaaagctga actgagtagt gccggtggag gcggctctgg tggcggaggt
     1441 agccatatga tctacctgaa atctttcgaa cgtaacatcc gcctgatcaa tatgaaagtg
     1501 gtttacgatg atgttcgtgt gctgaaagat attatccagg cgctggcccg cctggttgat
     1561 gaagccgtgc tgaaattcaa acaggatagc gttgaactgg tggcactgga tcgtgcgcat
     1621 attagcctga tctctgttaa cctgccgcgc gaaatgttca aagaatacga tgtgaacgat
     1681 gaatttaaat tcggtttcaa cacccagtac ctgatgaaaa tcctgaaagt tgcaaaacgt
     1741 aaagaagcca ttgaaatcgc aagtgaaagc ccggatagtg tgattatcaa cattatcggc
     1801 agcaccaatc gtgaatttaa cgtttgcaat ctggaagtgt ctgaacagga aattccggaa
     1861 atcaacctgc agttcgatat tagtgcgacg atcagctctg atggttttaa aagtgccatc
     1921 agcgaagttt ctaccgtgac ggataatgtg gttgtggaag gtcacgaaga tcgcattctg
     1981 atcaaagcag aaggcgaaag cgaagttgaa gtggaatttt ctaaagattg cggcggtctg
     2041 caggatctgg aatttagtaa agaaagcaaa aattcttaca gtgcggaata cctggatgat
     2101 gttctgtctc tgacgaaact gagtgattac gtgaaaatca gcttcggcaa ccagaaaccg
     2161 ctgcagctgt ttttcaatat ggaaggcggt ggcaaagtta cgtacctgct ggcgccgaaa
     2221 gttggcggta gcatgacgac tgaaaccata caaagcaacg ccaatcttgc ccctctgcca
     2281 ccccatgtgc cagagcacct ggtattcgac ttcgacatgt acaatccgtc gaatctgtct
     2341 gccggcgtgc aggaggcctg ggcagttctg caagaatcaa acgtaccgga tctggtgtgg
     2401 actcgctgca acggcggaca ctggatcgcc actcgcggcc aactgatccg tgaggcctat
     2461 gaagattacc gccacttttc cagcgagtgc ccgttcatcc ctcgtgaagc cggcgaagcc
     2521 tacgacttca ttcccacctc gatggacccg cccgagcagc gccagtttcg tgcgctggcc
     2581 aaccaagtgg ttggcatgcc ggtggtggat aagctggaga accgtatcca ggagctggcc
     2641 tgctcgctga tcgagagcct gcgcccgcaa ggacagtgca acttcaccga ggactacgcc
     2701 gaacccttcc cgatacgcat cttcatgctg ctcgcaggtc taccggaaga agatatcccg
     2761 cacttgaaat acctaacgga tcagatgacc cgtccggatg gcagcatgac cttcgcagag
     2821 gccaaggagg cgctctacga ctatctgata ccgatcatcg agcaacgcag gcagaagccg
     2881 ggaaccgacg ctatcagcat cgttgccaac ggccaggtca atgggcgacc gatcaccagt
     2941 gacgaagcca agaggatgtg tggcctgtta ctggtcggcg gcctggatac ggtggtcaat
     3001 ttcctcagct tcagcatgga gttcctggcc aaaagcccgg agcatcgcca ggagctgatc
     3061 gagcgtcccg agcgtattcc agccgcttgc gaggaactac tccggcgctt ctcgctggtt
     3121 gccgatggcc gcatcctcac ctccgattac gagtttcatg gcgtgcaact gaagaaaggt
     3181 gaccagatcc tgctaccgca gatgctgtct ggcctggatg agcgcgaaaa cgcctgcccg
     3241 atgcacgtcg acttcagtcg ccaaaaggtt tcacacacca cctttggcca cggcagccat
     3301 ctgtgccttg gccagcacct ggcccgccgg gaaatcatcg tcaccctcaa ggaatggctg
     3361 accaggattc ctgacttctc cattgccccg ggtgcccaga ttcagcacaa gagcggcatc
     3421 gtcagcggcg tgcaggcact ccctctggtc tgggacccgg cgactaccaa agcggtataa
     3481 ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     3541 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     3601 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     3661 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     3721 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3781 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3841 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3901 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3961 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4021 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4081 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4141 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4201 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     4261 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     4321 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     4381 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     4441 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     4501 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     4561 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     4621 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     4681 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     4741 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     4801 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     4861 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     4921 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     4981 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     5041 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     5101 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5161 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5221 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5281 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     5341 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5401 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5461 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5521 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5581 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5641 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5701 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5761 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5821 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5881 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5941 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6001 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     6061 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     6121 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     6181 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     6241 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     6301 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     6361 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     6421 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     6481 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     6541 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     6601 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     6661 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     6721 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     6781 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     6841 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     6901 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     6961 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     7021 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     7081 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     7141 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     7201 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     7261 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     7321 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     7381 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     7441 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     7501 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     7561 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     7621 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     7681 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     7741 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     7801 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     7861 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     7921 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     7981 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     8041 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     8101 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     8161 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     8221 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     8281 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     8341 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     8401 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     8461 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     8521 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     8581 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     8641 cgagatctcg atcccgcgaa at
//
