LOCUS       sequence-209048-        7348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MsePCNA1-PdR fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7348)
  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 7348)
  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: pM1R
FEATURES             Location/Qualifiers
     source          1..7348
                     /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             2213..2230
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2283..2301)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2297..2344
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2381..2836
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2468..2487)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2678..2699
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2928..3743)
                     /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(2928..3743)
                     /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     3651..3670
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3735..3754
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3865..4453
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4354..4373
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4607..4624
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4639..4778
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4704..4723)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4864..4886
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4880..5071)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5880..6962)
                     /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     6924..6943
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6963..7040)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7246..7265
                     /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 ttccgtgcaa tctatggtag ctctcgtgac
      181 ttctattaca tcgtgtctag cattagcaaa atcagcgacg agctgaccct gaacttcact
      241 gaagagggca tcggctccaa gtatctgacc gatgacaagg ttatgatggg tgtggttgaa
      301 attggtaaag acgctctgga agaatactct atcgagaagc cgatttctgt gaagctgaac
      361 ctgggcgaac tgaaaaagat cctgtccaag atgaagggcc gcagctccgt ggagattact
      421 gaaaccaacg aaggcatccg catcagcatg aaggacgaga aaaccggcac ccgctcctct
      481 ctgtccatca aggcggaaaa aggtgaaccg cagattctga aagagccgtc tgttgcgcac
      541 tctgttacta tgggtattgg cggtgacatc ctgagcatcc tggtggatga atccatgcag
      601 gttggcgagg aagtggaaat caaagcggag gatgaccacg tgagcttcga ggttgaggaa
      661 gctggcaaga aatactccgc ggtgctgaag aacggtaaac cgctgactaa actggaaatt
      721 gagaaacagg gctccagccg ttactccctg gcgattctgg agaaagttag cagcgcactg
      781 tccttctcca aagagattga gatcggtttc ggtgcgggta ttccgatgaa actgactgcg
      841 ccgctggaaa agggcgcagg cattcgtttc tgggttgctc cgcgtctggg aggtagcatg
      901 aacgcaaacg acaacgtggt gattgttggc actggtctgg ctggcgttga agttgcgttt
      961 ggtctgcgtg caagcggctg ggaaggtaac atccgcctgg tgggtgatgc gactgttatt
     1021 ccgcaccacc tgccgccgct gtctaaagcg tacctggcgg gcaaggcgac tgcggagtct
     1081 ctgtacctgc gcactccgga tgcttacgcg gctcagaaca tccagctgct gggcggtacc
     1141 caggttaccg cgattaaccg tgaccgtcag caggtgattc tgtctgatgg ccgtgcactg
     1201 gattatgatc gtctggtgct ggcaactggc ggtcgtccgc gtccgctgcc ggtggctagc
     1261 ggcgctgttg gtaaagcaaa caacttccgt tatctgcgta ccctggaaga cgcagaatgc
     1321 atccgtcgcc agctgattgc ggacaaccgt ctggttgtta tcggtggcgg ctacatcggt
     1381 ctggaggttg ctgctactgc gatcaaagcg aacatgcacg tgactctgct ggacactgct
     1441 gcgcgtgttc tggaacgtgt gactgctccg ccggtgagcg cattctacga gcatctgcac
     1501 cgcgaagctg gtgtggacat tcgtactggt actcaggttt gcggctttga aatgtctacc
     1561 gatcagcaga aagttactgc ggttctgtgc gaagacggca ctcgtctgcc ggctgatctg
     1621 gttattgcgg gtattggcct gatcccgaac tgcgaactgg catccgcagc gggcctgcag
     1681 gttgacaacg gcatcgtgat caacgagcac atgcagacta gcgatccgct gattatggca
     1741 gtgggcgatt gtgcgcgttt ccactctcag ctgtatgacc gttgggttcg catcgaaagc
     1801 gtgccgaacg cgctggaaca ggcgcgtaaa atcgcagcaa tcctgtgcgg taaagttccg
     1861 cgtgacgagg cggctccgtg gttttggagc gaccagtatg aaatcggcct gaaaatggtg
     1921 ggcctgtccg aaggttacga ccgtatcatc gttcgtggct ctctggcgca gccggacttc
     1981 tctgttttct atctgcaggg tgatcgcgtt ctggcagttg acactgtgaa ccgtccggtg
     2041 gaatttaacc agtctaaaca gatcattact gatcgcctgc cggttgagcc gaacctgctg
     2101 ggtgacgaat ctgtgccgct gaaggagatc atcgcggctg caaaagctga actgtcttct
     2161 gcgtgaggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca
     2221 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     2281 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     2341 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     2401 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2461 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2521 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2581 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2641 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2701 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2761 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2821 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2881 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     2941 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     3001 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     3061 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     3121 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     3181 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     3241 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     3301 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     3361 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     3421 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     3481 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     3541 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     3601 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     3661 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     3721 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     3781 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3841 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3901 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3961 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4021 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4081 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4141 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4201 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4261 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4321 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4381 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4441 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4501 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4561 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4621 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4681 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4741 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4801 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4861 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4921 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4981 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     5041 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5101 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5161 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5221 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5281 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5341 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5401 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5461 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5521 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5581 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5641 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     5701 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     5761 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5821 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5881 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5941 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     6001 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     6061 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     6121 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     6181 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     6241 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6301 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     6361 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     6421 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     6481 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6541 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     6601 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     6661 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     6721 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6781 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6841 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6901 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6961 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     7021 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     7081 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     7141 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     7201 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     7261 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     7321 gaggatcgag atctcgatcc cgcgaaat
//
