LOCUS       sequence-208518-        8166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses P450cam-PCNA3(R112C/T180C)-PdX fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8166)
  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 8166)
  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+C3(R112C/T180C)X
FEATURES             Location/Qualifiers
     source          1..8166
                     /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(2667..2685)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2681..2728
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2903..2908,2909..2925,2926..2931))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2958..2976)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3044..3148
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3149..3217,3218..4009)
                     /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(3367..3386)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4180..4768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4669..4688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4922..4939
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4954..5093
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5019..5038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5179..5201
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5195..5386)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6698..7780)
                     /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     7742..7761
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7781..7858)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8064..8083
                     /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 acgactgaaa ccatacaaag caacgccaat
      181 cttgcccctc tgccacccca tgtgccagag cacctggtat tcgacttcga catgtacaat
      241 ccgtcgaatc tgtctgccgg cgtgcaggag gcctgggcag ttctgcaaga atcaaacgta
      301 ccggatctgg tgtggactcg ctgcaacggc ggacactgga tcgccactcg cggccaactg
      361 atccgtgagg cctatgaaga ttaccgccac ttttccagcg agtgcccgtt catccctcgt
      421 gaagccggcg aagcctacga cttcattccc acctcgatgg acccgcccga gcagcgccag
      481 tttcgtgcgc tggccaacca agtggttggc atgccggtgg tggataagct ggagaaccgt
      541 atccaggagc tggcctgctc gctgatcgag agcctgcgcc cgcaaggaca gtgcaacttc
      601 accgaggact acgccgaacc cttcccgata cgcatcttca tgctgctcgc aggtctaccg
      661 gaagaagata tcccgcactt gaaataccta acggatcaga tgacccgtcc ggatggcagc
      721 atgaccttcg cagaggccaa ggaggcgctc tacgactatc tgataccgat catcgagcaa
      781 cgcaggcaga agccgggaac cgacgctatc agcatcgttg ccaacggcca ggtcaatggg
      841 cgaccgatca ccagtgacga agccaagagg atgtgtggcc tgttactggt cggcggcctg
      901 gatacggtgg tcaatttcct cagcttcagc atggagttcc tggccaaaag cccggagcat
      961 cgccaggagc tgatcgagcg tcccgagcgt attccagccg cttgcgagga actactccgg
     1021 cgcttctcgc tggttgccga tggccgcatc ctcacctccg attacgagtt tcatggcgtg
     1081 caactgaaga aaggtgacca gatcctgcta ccgcagatgc tgtctggcct ggatgagcgc
     1141 gaaaacgcct gcccgatgca cgtcgacttc agtcgccaaa aggtttcaca caccaccttt
     1201 ggccacggca gccatctgtg ccttggccag cacctggccc gccgggaaat catcgtcacc
     1261 ctcaaggaat ggctgaccag gattcctgac ttctccattg ccccgggtgc ccagattcag
     1321 cacaagagcg gcatcgtcag cggcgtgcag gcactccctc tggtctggga cccggcgact
     1381 accaaagcgg taggtggagg cggctctggt ggcggaggta gccatatgat ctacctgaaa
     1441 tctttcgaac gtaacatccg cctgatcaat atgaaagtgg tttacgatga tgttcgtgtg
     1501 ctgaaagata ttatccaggc gctggcccgc ctggttgatg aagccgtgct gaaattcaaa
     1561 caggatagcg ttgaactggt ggcactggat cgtgcgcata ttagcctgat ctctgttaac
     1621 ctgccgcgcg aaatgttcaa agaatacgat gtgaacgatg aatttaaatt cggtttcaac
     1681 acccagtacc tgatgaaaat cctgaaagtt gcaaaacgta aagaagccat tgaaatcgca
     1741 agtgaaagcc cggatagtgt gattatcaac attatcggca gcaccaatcg tgaatttaac
     1801 gtttgcaatc tggaagtgtc tgaacaggaa attccggaaa tcaacctgca gttcgatatt
     1861 agtgcgacga tcagctctga tggttttaaa agtgccatca gcgaagtttc taccgtgacg
     1921 gataatgtgg ttgtggaagg tcacgaagat cgcattctga tcaaagcaga aggcgaaagc
     1981 gaagttgaag tggaattttc taaagattgc ggcggtctgc aggatctgga atttagtaaa
     2041 gaaagcaaaa attcttacag tgcggaatac ctggatgatg ttctgtctct gacgaaactg
     2101 agtgattacg tgaaaatcag cttcggcaac cagaaaccgc tgcagctgtt tttcaatatg
     2161 gaaggcggtg gcaaagttac gtacctgctg gcgccgaaag tgggcggtgg cggcagccct
     2221 ccacctccac cgcctcctcc accgccacca ccgccgcctc cacctccacc gccgcccgga
     2281 ggtggtggct ctatgtctaa agtagtgtat gtgtcacatg atggaacgcg tcgcgaactg
     2341 gatgtggcgg atggcgtcag cctgatgcag gctgcagtct ccaatggtat ctacgatatt
     2401 gtcggtgatt gtggcggcag cgccagctgt gccacctgcc atgtctatgt gaacgaagcg
     2461 ttcacggaca aggtgcccgc cgccaacgag cgggaaatcg gcatgctgga gagcgtcacg
     2521 gccgaactga agccgaacag caggctcagc tgccagatca tcatgacgcc cgagctggat
     2581 ggcatcgtgg tcgatgttcc cgataggcaa tggtaaggat ccggctgcta acaaagcccg
     2641 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2701 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatatcccgc
     2761 aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg
     2821 ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa
     2881 tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa
     2941 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     3001 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3061 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3121 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3181 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3241 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3301 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3361 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     3421 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3481 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3541 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3601 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3661 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     3721 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3781 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3841 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3901 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3961 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4021 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4081 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4141 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4201 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4261 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4321 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4381 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4441 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4501 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4561 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4621 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4681 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4741 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4801 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4861 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4921 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4981 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     5041 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     5101 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5161 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5221 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     5281 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     5341 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     5401 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     5461 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     5521 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     5581 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     5641 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     5701 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     5761 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     5821 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     5881 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     5941 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     6001 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     6061 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     6121 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     6181 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     6241 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     6301 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     6361 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     6421 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     6481 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     6541 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     6601 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     6661 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6721 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6781 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     6841 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     6901 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     6961 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     7021 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     7081 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     7141 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     7201 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     7261 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     7321 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     7381 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     7441 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     7501 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     7561 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     7621 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     7681 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     7741 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     7801 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     7861 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     7921 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     7981 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     8041 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     8101 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     8161 cgaaat
//
