LOCUS       sequence-209049-        7321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MsePCNA3-CYP101A1 fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7321)
  AUTHORS   Iwata F, Hirakawa H, Nagamune T
  TITLE     A Stable Artificial Multienzymatic Complex Using a Heterotrimeric 
            Protein From Metallosphaera sedula.
  JOURNAL   Biotechnol J. 2018 Nov;13(11):e1700662. doi: 10.1002/biot.201700662.
            Epub 2018 Apr 30.
  PUBMED    29663675
REFERENCE   2  (bases 1 to 7321)
  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: "2018-11"; volume: "13(11):e1700662. doi"; pages: " 
            10.1002/biot.201700662. Epub 2018 Apr 30"
COMMENT     Sequence Label: pM3C
FEATURES             Location/Qualifiers
     source          1..7321
                     /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             2186..2203
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2256..2274)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2270..2317
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2354..2809
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2441..2460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2651..2672
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2901..3716)
                     /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(2901..3716)
                     /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     3624..3643
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3708..3727
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3838..4426
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4327..4346
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4580..4597
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4612..4751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4677..4696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4837..4859
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4853..5044)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5853..6935)
                     /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     6897..6916
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6936..7013)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7219..7238
                     /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 cgtatcgcat acgcgaacgc gatggacttc
      181 aaaaccgtga tcgaagcgct gtctaaactg atcgacgaag tgaccttcac tttcaccagc
      241 tctggtctgg acgttgttgc ggttgaccgt gcgcatatca gcctgatcaa gctgcacttc
      301 ccgaaagaag cgttcgaaga atttgacgtg gaagaccagt tccgtttcgg ctttaacacc
      361 cagtacatgc tgaaggttat ggcgtctgcg aaacgcaagg agaagattga gatggagtcc
      421 cgtgaagagt ccgaaatcgt tatccgtatg ctgggcgaac cgccgcgtga atttaccatc
      481 cgtaacattg aggttccgat ccaggaactg ccggagctga aactggattt cgacgtgaag
      541 gcgaaaatca cttctggcgg cttcaagaag gctgttagcg agattgcgac cgtttctgac
      601 tctgtggaga ttgacgcgac cgaaatggag attaagctgc gttccaagga gtccaccgag
      661 atcgaggtgg agttctccaa agaaatgggt ggtctgcagg aaattgaggt gaagaagccg
      721 tctgtttctt cctacccgtc tgactacctg gaggacgttc tggttctgac ccgcctgtct
      781 ggcttcctga acctgctgtt ctctgagcag aaaccgctgc agctggagtt caacatggac
      841 aacggtggct ccgtggttta tctgctggcg ccgaacgttg gtggaggtag catgactacc
      901 gaaaccatcc agtctaacgc gaacctggct ccgctgccgc cgcatgtgcc ggaacacctg
      961 gtgttcgact tcgatatgta caacccgtct aacctgtctg cgggcgttca ggaagcgtgg
     1021 gcggtgctgc aggaatccaa cgttccggat ctggtgtgga cccgctgcaa cggcggtcac
     1081 tggatcgcga cccgtggcca gctgattcgt gaagcatacg aagattaccg tcatttctcc
     1141 tctgaatgcc cgttcatccc gcgtgaggct ggtgaggcgt acgatttcat tccgactagc
     1201 atggacccgc cggagcagcg ccagtttcgt gcgctggcga accaggttgt tggtatgccg
     1261 gttgtggata aactggagaa ccgtatccag gagctggcgt gctctctgat tgagtctctg
     1321 cgtccgcagg gtcagtgcaa cttcaccgaa gactacgcgg aaccgtttcc gatccgtatt
     1381 ttcatgctgc tggcgggtct gccggaggaa gacattccgc acctgaagta tctgaccgac
     1441 cagatgactc gcccggatgg ctctatgacc ttcgcggagg cgaaagaagc gctgtacgac
     1501 tacctgatcc cgatcatcga acagcgccgt cagaagccgg gtaccgacgc gatctctatc
     1561 gttgcgaacg gtcaggttaa cggtcgtccg attacctccg atgaagcgaa acgtatgtgc
     1621 ggtctgctgc tggtgggtgg tctggacact gttgttaact tcctgtcttt ctctatggaa
     1681 tttctggcta aaagcccgga acatcgtcag gaactgatcg agcgtccgga gcgcattccg
     1741 gctgcgtgcg aagaactgct gcgtcgcttc tctctggttg cagacggtcg catcctgacc
     1801 tctgactacg agttccacgg tgtgcagctg aaaaaaggtg atcagatcct gctgccgcag
     1861 atgctgagcg gcctggacga acgtgaaaac gcgtgtccga tgcacgttga cttttctcgc
     1921 cagaaggttt ctcacactac tttcggtcac ggctctcacc tgtgcctggg ccagcacctg
     1981 gcgcgtcgcg agatcattgt taccctgaaa gaatggctga ctcgtatccc ggatttcagc
     2041 attgcaccgg gtgcgcagat ccagcacaaa tctggtatcg ttagcggcgt gcaggctctg
     2101 ccgctggttt gggacccggc aaccactaaa gcggtttgag gatccgaatt cgagctccgt
     2161 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     2221 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     2281 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2341 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2401 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2461 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2521 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2581 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2641 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2701 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2761 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     2821 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2881 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     2941 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     3001 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     3061 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     3121 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     3181 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     3241 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     3301 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     3361 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     3421 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     3481 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     3541 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     3601 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     3661 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     3721 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     3781 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3841 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3901 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3961 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4021 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4081 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4141 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4201 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4261 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4321 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4381 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4441 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4501 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4561 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4621 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     4681 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4741 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4801 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4861 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4921 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4981 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     5041 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     5101 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     5161 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     5221 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     5281 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     5341 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     5401 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     5461 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     5521 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     5581 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     5641 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     5701 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     5761 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     5821 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5881 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5941 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     6001 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     6061 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     6121 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     6181 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     6241 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     6301 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     6361 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     6421 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     6481 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     6541 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     6601 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     6661 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     6721 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     6781 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     6841 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     6901 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6961 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     7021 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     7081 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     7141 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     7201 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     7261 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     7321 t
//
