LOCUS       sequence-208580-        6849 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 6849)
  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 6849)
  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+2(L171C)X
FEATURES             Location/Qualifiers
     source          1..6849
                     /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(1350..1368)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1364..1411
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1586..1591,1592..1608,1609..1614))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1641..1659)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1727..1831
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1832..1900,1901..2692)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2050..2069)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2863..3451
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3352..3371
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3605..3622
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3637..3776
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3702..3721)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3862..3884
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3878..4069)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5381..6463)
                     /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     6425..6444
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6464..6541)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6747..6766
                     /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 atgaaagcca aagtgatcga tgcagtttct
      181 ttcagttaca tcctgcgtac cgtgggcgat ttcctgagcg aagccaactt tattgtgacg
      241 aaagaaggca ttcgtgttag tggtatcgat ccgagccgcg tggtttttct ggatatcttc
      301 ctgccgagct cttactttga aggcttcgaa gttagccagg aaaaagaaat catcggtttc
      361 aaactggaag atgtgaacga tattctgaaa cgtgttctga aagatgatac cctgatcctg
      421 agtagcaatg aatctaaact gaccctgacg tttgatggtg aatttacccg cagttttgaa
      481 ctgccgctga ttcaggtgga atctacgcag ccgccgagtg ttaatctgga atttccgttc
      541 aaagcgcagc tgctgaccat tacgtttgcc gatattatcg atgaactgag tgatctgggc
      601 gaagtgctga acatccatag caaagaaaac aaactgtact tcgaagtgat cggtgattgc
      661 agcaccgcga aagttgaact gtctaccgat aacggcacgc tgctggaagc gtctggtgcc
      721 gatgtgtcta gtagctatgg catggaatac gttgcaaata ccacgaaaat gcgtcgcgcg
      781 agcgattcta tggaactgta cttcggtagc cagattccgc tgaaactgcg ttttaaactg
      841 ccgcaggaag gctacggtga tttctacatc gcaccgcgcg cggacggcgg tggcggcagc
      901 cctccacctc caccgcctcc tccaccgcca ccaccgccgc ctccacctcc accgccgccc
      961 ggaggtggtg gctctatgtc taaagtagtg tatgtgtcac atgatggaac gcgtcgcgaa
     1021 ctggatgtgg cggatggcgt cagcctgatg caggctgcag tctccaatgg tatctacgat
     1081 attgtcggtg attgtggcgg cagcgccagc tgtgccacct gccatgtcta tgtgaacgaa
     1141 gcgttcacgg acaaggtgcc cgccgccaac gagcgggaaa tcggcatgct ggagagcgtc
     1201 acggccgaac tgaagccgaa cagcaggctc agctgccaga tcatcatgac gcccgagctg
     1261 gatggcatcg tggtcgatgt tcccgatagg caatggtaag gatccggctg ctaacaaagc
     1321 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1381 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc
     1441 cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg
     1501 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag
     1561 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga
     1621 gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     1681 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1741 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     1801 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     1861 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     1921 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1981 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     2041 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     2101 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2161 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     2221 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     2281 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     2341 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     2401 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     2461 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     2521 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     2581 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     2641 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     2701 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     2761 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     2821 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2881 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2941 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3001 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3061 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3121 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3181 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3241 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3301 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3361 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3421 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3481 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3541 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3601 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3661 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3721 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3781 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3841 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3901 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3961 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4021 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4081 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4141 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4201 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4261 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4321 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4381 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4441 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4501 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     4561 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     4621 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     4681 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     4741 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     4801 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt
     4861 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     4921 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     4981 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     5041 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     5101 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     5161 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     5221 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     5281 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     5341 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5401 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5461 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     5521 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     5581 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5641 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     5701 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5761 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5821 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5881 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5941 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     6001 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     6061 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     6121 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     6181 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     6241 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     6301 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     6361 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     6421 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     6481 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     6541 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6601 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6661 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6721 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6781 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     6841 ccgcgaaat
//
