LOCUS       sequence-209046-        7363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 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 7363)
  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 7363)
  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+3(R112C/T180C)C
FEATURES             Location/Qualifiers
     source          1..7363
                     /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             2228..2245
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2298..2316)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2312..2359
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2396..2851
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2483..2502)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2693..2714
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2943..3758)
                     /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(2943..3758)
                     /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     3666..3685
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3750..3769
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3880..4468
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4369..4388
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4622..4639
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4654..4793
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4719..4738)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4879..4901
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4895..5086)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5895..6977)
                     /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     6939..6958
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6978..7055)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7261..7280
                     /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 atctacctga aatctttcga acgtaacatc
      181 cgcctgatca atatgaaagt ggtttacgat gatgttcgtg tgctgaaaga tattatccag
      241 gcgctggccc gcctggttga tgaagccgtg ctgaaattca aacaggatag cgttgaactg
      301 gtggcactgg atcgtgcgca tattagcctg atctctgtta acctgccgcg cgaaatgttc
      361 aaagaatacg atgtgaacga tgaatttaaa ttcggtttca acacccagta cctgatgaaa
      421 atcctgaaag ttgcaaaacg taaagaagcc attgaaatcg caagtgaaag cccggatagt
      481 gtgattatca acattatcgg cagcaccaat cgtgaattta acgtttgcaa tctggaagtg
      541 tctgaacagg aaattccgga aatcaacctg cagttcgata ttagtgcgac gatcagctct
      601 gatggtttta aaagtgccat cagcgaagtt tctaccgtga cggataatgt ggttgtggaa
      661 ggtcacgaag atcgcattct gatcaaagca gaaggcgaaa gcgaagttga agtggaattt
      721 tctaaagatt gcggcggtct gcaggatctg gaatttagta aagaaagcaa aaattcttac
      781 agtgcggaat acctggatga tgttctgtct ctgacgaaac tgagtgatta cgtgaaaatc
      841 agcttcggca accagaaacc gctgcagctg tttttcaata tggaaggcgg tggcaaagtt
      901 acgtacctgc tggcgccgaa agttggcggt agcatgacga ctgaaaccat acaaagcaac
      961 gccaatcttg cccctctgcc accccatgtg ccagagcacc tggtattcga cttcgacatg
     1021 tacaatccgt cgaatctgtc tgccggcgtg caggaggcct gggcagttct gcaagaatca
     1081 aacgtaccgg atctggtgtg gactcgctgc aacggcggac actggatcgc cactcgcggc
     1141 caactgatcc gtgaggccta tgaagattac cgccactttt ccagcgagtg cccgttcatc
     1201 cctcgtgaag ccggcgaagc ctacgacttc attcccacct cgatggaccc gcccgagcag
     1261 cgccagtttc gtgcgctggc caaccaagtg gttggcatgc cggtggtgga taagctggag
     1321 aaccgtatcc aggagctggc ctgctcgctg atcgagagcc tgcgcccgca aggacagtgc
     1381 aacttcaccg aggactacgc cgaacccttc ccgatacgca tcttcatgct gctcgcaggt
     1441 ctaccggaag aagatatccc gcacttgaaa tacctaacgg atcagatgac ccgtccggat
     1501 ggcagcatga ccttcgcaga ggccaaggag gcgctctacg actatctgat accgatcatc
     1561 gagcaacgca ggcagaagcc gggaaccgac gctatcagca tcgttgccaa cggccaggtc
     1621 aatgggcgac cgatcaccag tgacgaagcc aagaggatgt gtggcctgtt actggtcggc
     1681 ggcctggata cggtggtcaa tttcctcagc ttcagcatgg agttcctggc caaaagcccg
     1741 gagcatcgcc aggagctgat cgagcgtccc gagcgtattc cagccgcttg cgaggaacta
     1801 ctccggcgct tctcgctggt tgccgatggc cgcatcctca cctccgatta cgagtttcat
     1861 ggcgtgcaac tgaagaaagg tgaccagatc ctgctaccgc agatgctgtc tggcctggat
     1921 gagcgcgaaa acgcctgccc gatgcacgtc gacttcagtc gccaaaaggt ttcacacacc
     1981 acctttggcc acggcagcca tctgtgcctt ggccagcacc tggcccgccg ggaaatcatc
     2041 gtcaccctca aggaatggct gaccaggatt cctgacttct ccattgcccc gggtgcccag
     2101 attcagcaca agagcggcat cgtcagcggc gtgcaggcac tccctctggt ctgggacccg
     2161 gcgactacca aagcggtata aggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc
     2221 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     2281 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2341 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     2401 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2461 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2521 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2581 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2641 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2701 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2761 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2821 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     2881 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2941 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     3001 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     3061 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     3121 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     3181 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     3241 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     3301 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     3361 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     3421 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     3481 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3541 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3601 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     3661 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3721 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     3781 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     3841 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3901 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3961 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4021 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4081 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4141 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4201 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4261 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4321 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4381 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4441 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4501 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4561 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4621 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4681 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     4741 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     4801 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4861 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4921 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4981 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     5041 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     5101 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     5161 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     5221 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     5281 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     5341 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     5401 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     5461 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     5521 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     5581 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     5641 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     5701 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     5761 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5821 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5881 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5941 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     6001 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     6061 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     6121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     6181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     6241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     6301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     6361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     6481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6961 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     7021 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     7081 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     7141 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     7201 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     7261 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     7321 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aat
//
