LOCUS       sequence-209051-        6480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MSePCNA2-PdX fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6480)
  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 6480)
  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: pM2P20X
FEATURES             Location/Qualifiers
     source          1..6480
                     /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             1345..1362
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1415..1433)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1429..1476
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1513..1968
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1600..1619)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1810..1831
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2060..2875)
                     /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(2060..2875)
                     /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     2783..2802
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2867..2886
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2997..3585
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3486..3505
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3739..3756
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3771..3910
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3836..3855)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3996..4018
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4012..4203)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5012..6094)
                     /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     6056..6075
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6095..6172)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6378..6397
                     /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 aaattcaaag ttatcgacgc gaactctatg
      181 gcgaccatct tccgtaccat cggcgagttt atgccggaga ttaccctgat cggtaccaaa
      241 gaaggtgttc gtctgagcgg tgttgacccg gcacgtgttg cgctgatcga catcttcatc
      301 ccgcaggcgt acttccacga gtacgaaagc gcagagaaag aaaccgttac cgtgaagctg
      361 gaggagatca tcgcgagcct gaaaaacgtg aagaagaacg actctctgac cttccagtct
      421 ggtgaggacc gtctgatgat cactctggac ggtgactttg agcgcacctt ttatctgccg
      481 atcctgatgg gcgaggaacc gtctctgccg tccatcaaac tggagttcgc gttcaaggcg
      541 aaaatgctga cctctacctt ttccaacgtt atgcagatcc tgggcgacct gggtgacgcg
      601 ctgaccctgt ctgcggaggg tggtaaactg actttcatgg ttgagggcga cgttggttct
      661 agcaaggtgg agctgtccga agaatccggt actctgctgg aagcgaccgg tgcggacgct
      721 aagggcacct acggcatgga ctacctggtt aagaccgcga agatgcgcaa ctcttccgac
      781 atcgttgaac tgatgtttgg ttcccagctg ccgattaagc tgcgtttcga actgccgcag
      841 gaaggctacg gcgacttcta tattgcaccg cgtgcggaag gaggtggcgg ttcccctcca
      901 cctccaccgc ctcctccacc gccaccaccg ccgcctccac ctccaccgcc gcccggcgga
      961 ggtggctcta tgtctaaagt tgtttacgtt tctcacgacg gtacccgtcg cgaactggac
     1021 gttgcggacg gtgtttctct gatgcaggcg gcagtttcta acggcatcta cgacatcgtt
     1081 ggtgactgcg gtggttctgc gtcttgcgcg acctgccacg tgtacgtgaa cgaagcgttc
     1141 accgacaaag ttccggctgc taacgaacgt gaaatcggta tgctggaatc tgttaccgcg
     1201 gaactgaaac cgaactctcg tctgtcttgc cagatcatca tgaccccgga gctggacggt
     1261 atcgttgttg acgttccgga ccgtcagtgg tgagaattcg atccgaattc gagctccgtc
     1321 gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
     1381 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1441 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1501 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1561 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1621 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1681 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1741 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1801 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1861 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1921 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1981 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2041 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2101 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2161 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2221 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2281 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2341 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2401 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2461 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2521 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2581 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2641 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2701 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2761 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2821 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2881 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     2941 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3001 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3061 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3121 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3181 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3241 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3301 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3361 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3421 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3481 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3541 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3601 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3661 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3721 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3781 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3841 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3901 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3961 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4021 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4081 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     4141 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4201 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4261 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4321 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4381 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4441 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4501 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4561 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4621 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     4681 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4741 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4801 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4861 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4921 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     4981 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5041 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5101 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     5161 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     5221 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5281 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5341 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5401 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5461 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5521 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5581 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5641 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5701 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5761 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5821 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5881 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     5941 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     6001 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     6061 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     6121 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6181 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6241 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6301 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6361 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6421 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
