LOCUS       sequence-208598-        7408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PdR-PCNA3(R112C/T180C) fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7408)
  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 7408)
  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)
FEATURES             Location/Qualifiers
     source          1..7408
                     /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             2273..2290
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2343..2361)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2357..2404
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2441..2896
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2528..2547)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2738..2759
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2988..3803)
                     /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(2988..3803)
                     /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     3711..3730
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3795..3814
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3925..4513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4414..4433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4667..4684
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4699..4838
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4764..4783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4924..4946
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4940..5131)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5940..7022)
                     /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     6984..7003
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7023..7100)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7306..7325
                     /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 gtgtaaggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca
     2281 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     2341 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     2401 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     2461 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2521 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2581 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2641 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2701 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2761 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2821 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2881 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2941 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     3001 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     3061 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     3121 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     3181 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     3241 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     3301 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     3361 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     3421 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     3481 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     3541 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     3601 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     3661 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     3721 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     3781 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     3841 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3901 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3961 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4021 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4081 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4141 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4201 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4261 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4321 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4381 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4441 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4501 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4561 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4621 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4681 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4741 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4801 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4861 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4921 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4981 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     5041 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     5101 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5161 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5221 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5281 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5341 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5401 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5461 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5521 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5581 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5641 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5701 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     5761 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     5821 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5881 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5941 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6001 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     6061 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     6121 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     6181 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     6241 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     6301 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6361 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     6421 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     6481 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     6541 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6601 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     6661 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     6721 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     6781 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6841 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6901 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6961 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     7021 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     7081 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     7141 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     7201 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     7261 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     7321 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     7381 gaggatcgag atctcgatcc cgcgaaat
//
