LOCUS       sequence-208577-        8148 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PdR-PCNA2(L171C)-PdX fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8148)
  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 8148)
  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: pET15b+R2(L171C)X
FEATURES             Location/Qualifiers
     source          1..8148
                     /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"
     primer_bind     complement(2649..2667)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2663..2710
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2885..2890,2891..2907,2908..2913))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2940..2958)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3026..3130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3131..3199,3200..3991)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGQKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3349..3368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4162..4750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4651..4670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4904..4921
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4936..5075
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5001..5020)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5161..5183
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5177..5368)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6680..7762)
                     /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     7724..7743
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7763..7840)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8046..8065
                     /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 tgaaagccaa agtgatcgat gcagtttctt tcagttacat cctgcgtacc
     1501 gtgggcgatt tcctgagcga agccaacttt attgtgacga aagaaggcat tcgtgttagt
     1561 ggtatcgatc cgagccgcgt ggtttttctg gatatcttcc tgccgagctc ttactttgaa
     1621 ggcttcgaag ttagccagga aaaagaaatc atcggtttca aactggaaga tgtgaacgat
     1681 attctgaaac gtgttctgaa agatgatacc ctgatcctga gtagcaatga atctaaactg
     1741 accctgacgt ttgatggtga atttacccgc agttttgaac tgccgctgat tcaggtggaa
     1801 tctacgcagc cgccgagtgt taatctggaa tttccgttca aagcgcagct gctgaccatt
     1861 acgtttgccg atattatcga tgaactgagt gatctgggcg aagtgctgaa catccatagc
     1921 aaagaaaaca aactgtactt cgaagtgatc ggtgattgca gcaccgcgaa agttgaactg
     1981 tctaccgata acggcacgct gctggaagcg tctggtgccg atgtgtctag tagctatggc
     2041 atggaatacg ttgcaaatac cacgaaaatg cgtcgcgcga gcgattctat ggaactgtac
     2101 ttcggtagcc agattccgct gaaactgcgt tttaaactgc cgcaggaagg ctacggtgat
     2161 ttctacatcg caccgcgcgc ggacggcggt ggcggcagcc ctccacctcc accgcctcct
     2221 ccaccgccac caccgccgcc tccacctcca ccgccgcccg gaggtggtgg ctctatgtct
     2281 aaagtagtgt atgtgtcaca tgatggaacg cgtcgcgaac tggatgtggc ggatggcgtc
     2341 agcctgatgc aggctgcagt ctccaatggt atctacgata ttgtcggtga ttgtggcggc
     2401 agcgccagct gtgccacctg ccatgtctat gtgaacgaag cgttcacgga caaggtgccc
     2461 gccgccaacg agcgggaaat cggcatgctg gagagcgtca cggccgaact gaagccgaac
     2521 agcaggctca gctgccagat catcatgacg cccgagctgg atggcatcgt ggtcgatgtt
     2581 cccgataggc aatggtaagg atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2641 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     2701 gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc
     2761 ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc
     2821 gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta
     2881 ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa gacgaaaggg
     2941 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3001 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3061 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3121 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3181 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3241 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3301 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3361 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3421 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3481 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3541 gacaacgatc ggaggacaga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3601 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3661 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3721 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3781 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3841 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3901 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3961 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4021 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4081 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4741 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4801 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4861 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4921 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4981 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     5041 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     5101 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5161 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     5221 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     5281 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     5341 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     5401 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     5461 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     5521 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     5581 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     5641 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     5701 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     5761 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     5821 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg
     5881 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
     5941 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
     6001 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
     6061 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
     6121 ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
     6181 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
     6241 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
     6301 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
     6361 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     6421 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     6481 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     6541 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     6601 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     6661 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     6721 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     6781 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     6841 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     6901 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     6961 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     7021 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     7081 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     7141 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     7201 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     7261 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     7321 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     7381 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     7441 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     7501 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     7561 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     7621 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     7681 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     7741 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     7801 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     7861 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     7921 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     7981 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     8041 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     8101 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
