LOCUS       sequence-208514-        6917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PCNA1(G108C)-PdX fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6917)
  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 6917)
  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+1(G108C)X
FEATURES             Location/Qualifiers
     source          1..6917
                     /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(1359..1377)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1373..1420
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1595..1600,1601..1617,1618..1623))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1650..1668)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1736..1840
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1841..1909,1910..2701)
                     /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(2059..2078)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2872..3460
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3361..3380
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3614..3631
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3705..3844
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3770..3789)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3930..3952
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3946..4137)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5449..6531)
                     /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     6493..6512
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6532..6609)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6815..6834
                     /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 tttaaaattg tgtatccgaa cgccaaagat
      181 ttctttagct tcatcaacag cattaccaac gttacggatt ctatcatcct gaacttcacc
      241 gaagatggca tcttcagtcg tcatctgacg gaagataaag tgctgatggc aatcatgcgc
      301 attccgaaag atgttctgtc tgaatacagt attgatagcc cgacctctgt gaaactggat
      361 gtgagctctg tgaagaaaat tctgagcaaa gcaagtagca aaaaagcgac gattgaactg
      421 accgaaacgg attctggcct gaaaatcatc atccgtgatg aaaaatcttg cgccaaaagt
      481 accatctaca tcaaagcaga aaaaggccag gttgaacagc tgacggaacc gaaagtgaac
      541 ctggccgtta attttaccac ggatgaaagc gtgctgaacg ttatcgcggc cgatgtgacc
      601 ctggttggtg aagaaatgcg tatcagcacg gaagaagata aaatcaaaat tgaagcgggc
      661 gaagaaggta aacgctatgt ggcctttctg atgaaagata aaccgctgaa agaactgagc
      721 atcgatacct ctgcctctag tagctacagc gcagaaatgt tcaaagatgc ggtgaaaggc
      781 ctgcgtggtt ttagtgcgcc gacgatggtg tctttcggtg aaaatctgcc gatgaaaatt
      841 gatgtggaag cagttagcgg cggtcacatg atcttctgga ttgcgccgcg cttaggcggt
      901 ggcggcagcc ctccacctcc accgcctcct ccaccgccac caccgccgcc tccacctcca
      961 ccgccgcccg gaggtggtgg ctctatgtct aaagtagtgt atgtgtcaca tgatggaacg
     1021 cgtcgcgaac tggatgtggc ggatggcgtc agcctgatgc aggctgcagt ctccaatggt
     1081 atctacgata ttgtcggtga ttgtggcggc agcgccagct gtgccacctg ccatgtctat
     1141 gtgaacgaag cgttcacgga caaggtgccc gccgccaacg agcgggaaat cggcatgctg
     1201 gagagcgtca cggccgaact gaagccgaac agcaggctca gctgccagat catcatgacg
     1261 cccgagctgg atggcatcgt ggtcgatgtt cccgataggc aatggtaagg atccggctgc
     1321 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1381 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1441 cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc
     1501 agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga
     1561 ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt
     1621 caaacatgag aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     1681 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     1741 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1801 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1861 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1921 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1981 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     2041 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     2101 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     2161 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2221 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2281 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2341 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     2401 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2461 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2521 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2581 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2641 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2701 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2761 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2821 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2881 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2941 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3001 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3061 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3121 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3181 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3241 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3301 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3361 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3421 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3481 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3541 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3601 aacgaccgag cgcagcgagt cagtgagcga ggaagcggtt gagcgcctga tgcgagcgag
     3661 gaagcggttg agcgcctgat gcggagcgag gaagcggttg agcgcctgat gcggtatttt
     3721 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3781 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3841 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3901 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3961 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     4021 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     4081 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     4141 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     4201 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     4261 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     4321 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     4381 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4441 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4501 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4561 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
     4621 gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca
     4681 agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt
     4741 ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat
     4801 gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc
     4861 aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg
     4921 atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca
     4981 tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga
     5041 agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc
     5101 agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     5161 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     5221 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     5281 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     5341 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5401 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5461 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5521 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5581 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     5641 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     5701 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     5761 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     5821 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     5881 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     5941 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6001 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6061 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6121 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6181 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6241 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6301 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6361 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6421 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6481 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6541 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     6601 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     6661 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     6721 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     6781 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     6841 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     6901 tctcgatccc gcgaaat
//
