LOCUS       sequence-208597-        7737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PdR-PCNA2(L171C) fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7737)
  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 7737)
  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)
FEATURES             Location/Qualifiers
     source          1..7737
                     /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(2238..2256)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2252..2299
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2474..2479,2480..2496,2497..2502))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2529..2547)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2615..2719
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2720..2788,2789..3580)
                     /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(2938..2957)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3751..4339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4240..4259
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4493..4510
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4525..4664
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4590..4609)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4750..4772
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4766..4957)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6269..7351)
                     /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     7313..7332
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7352..7429)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7635..7654
                     /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 ggactaagga tccggctgct aacaaagccc gaaaggaagc
     2221 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2281 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc
     2341 ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg
     2401 acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt
     2461 gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag
     2521 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2581 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2641 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2701 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2761 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2821 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2881 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2941 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     3001 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3061 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3121 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3181 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3241 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3301 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3361 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3421 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3481 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3541 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3601 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3661 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3721 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3781 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3841 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3901 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3961 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4021 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4081 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4141 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4201 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4261 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4321 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4381 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4441 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4501 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4561 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4621 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4681 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4741 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4801 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4861 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4921 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4981 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5041 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5101 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5161 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5221 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5281 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5341 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5401 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc
     5461 ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca
     5521 cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt
     5581 gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg
     5641 gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc
     5701 cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag
     5761 agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat
     5821 ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc
     5881 catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc
     5941 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     6001 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     6061 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     6121 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     6181 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     6241 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6301 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6361 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     6421 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6481 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6541 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6601 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6661 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6721 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6781 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6841 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6901 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6961 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     7021 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     7081 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     7141 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     7201 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     7261 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     7321 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     7381 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     7441 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7501 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     7561 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     7621 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     7681 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
