LOCUS       sequence-208572-        6944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCNA1-PdX fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6944)
  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 6944)
  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: pET15T+1X
FEATURES             Location/Qualifiers
     source          1..6944
                     /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             151..171
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1386..1404)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1400..1447
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1622..1627,1628..1644,1645..1650))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1677..1695)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1763..1867
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1868..1936,1937..2728)
                     /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(2086..2105)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2899..3487
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3388..3407
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3641..3658
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3732..3871
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3797..3816)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3957..3979
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3973..4164)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5476..6558)
                     /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     6520..6539
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6559..6636)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6842..6861
                     /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 cagccatagc gaaaacctgt atttccaggg ccatatgttt
      181 aaaattgtgt atccgaacgc caaagatttc tttagcttca tcaacagcat taccaacgtt
      241 acggattcta tcatcctgaa cttcaccgaa gatggcatct tcagtcgtca tctgacggaa
      301 gataaagtgc tgatggcaat catgcgcatt ccgaaagatg ttctgtctga atacagtatt
      361 gatagcccga cctctgtgaa actggatgtg agctctgtga agaaaattct gagcaaagca
      421 agtagcaaaa aagcgacgat tgaactgacc gaaacggatt ctggcctgaa aatcatcatc
      481 cgtgatgaaa aatctggtgc caaaagtacc atctacatca aagcagaaaa aggccaggtt
      541 gaacagctga cggaaccgaa agtgaacctg gccgttaatt ttaccacgga tgaaagcgtg
      601 ctgaacgtta tcgcggccga tgtgaccctg gttggtgaag aaatgcgtat cagcacggaa
      661 gaagataaaa tcaaaattga agcgggcgaa gaaggtaaac gctatgtggc ctttctgatg
      721 aaagataaac cgctgaaaga actgagcatc gatacctctg cctctagtag ctacagcgca
      781 gaaatgttca aagatgcggt gaaaggcctg cgtggtttta gtgcgccgac gatggtgtct
      841 ttcggtgaaa atctgccgat gaaaattgat gtggaagcag ttagcggcgg tcacatgatc
      901 ttctggattg cgccgcgctt aggcggtggc ggcagccctc cacctccacc gcctcctcca
      961 ccgccaccac cgccgcctcc acctccaccg ccgcccggag gtggtggctc tatgtctaaa
     1021 gtagtgtatg tgtcacatga tggaacgcgt cgcgaactgg atgtggcgga tggcgtcagc
     1081 ctgatgcagg ctgcagtctc caatggtatc tacgatattg tcggtgattg tggcggcagc
     1141 gccagctgtg ccacctgcca tgtctatgtg aacgaagcgt tcacggacaa ggtgcccgcc
     1201 gccaacgagc gggaaatcgg catgctggag agcgtcacgg ccgaactgaa gccgaacagc
     1261 aggctcagct gccagatcat catgacgccc gagctggatg gcatcgtggt cgatgttccc
     1321 gataggcaat ggtaaggatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1381 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1441 ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc
     1501 ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca
     1561 ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg
     1621 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
     1681 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     1741 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1801 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     1861 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     1921 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     1981 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2041 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     2101 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     2161 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     2221 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     2281 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     2341 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     2401 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     2461 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     2521 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2581 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2641 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2701 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2761 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2821 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2881 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2941 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3001 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3061 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3121 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3181 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3241 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3301 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3361 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3421 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3481 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3541 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3601 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3661 agcggttgag cgcctgatgc gagcgaggaa gcggttgagc gcctgatgcg gagcgaggaa
     3721 gcggttgagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3781 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3841 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3901 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3961 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     4021 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     4081 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4141 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4201 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4261 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4321 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4381 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4441 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4501 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4561 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4621 gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat
     4681 ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa
     4741 gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc
     4801 attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg
     4861 tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
     4921 atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat
     4981 ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg
     5041 ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc
     5101 gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc
     5161 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     5221 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     5281 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     5341 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     5401 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     5461 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5521 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     5581 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5641 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5701 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     5761 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5821 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5881 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5941 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     6001 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     6061 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     6121 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     6181 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     6241 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     6301 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     6361 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     6421 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     6481 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     6541 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     6601 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     6661 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     6721 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6781 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6841 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6901 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat
//
