LOCUS       sequence-208655-        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SsoPCNA1-PdR fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  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 7354)
  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: pS1R
FEATURES             Location/Qualifiers
     source          1..7354
                     /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             2219..2236
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2289..2307)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2303..2350
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2387..2842
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2474..2493)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2684..2705
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2934..3749)
                     /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(2934..3749)
                     /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     3657..3676
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3741..3760
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3871..4459
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4360..4379
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4613..4630
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4645..4784
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4710..4729)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4870..4892
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4886..5077)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5886..6968)
                     /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     6930..6949
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6969..7046)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7252..7271
                     /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 aaaaatctgg tgccaaaagt
      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 cctgggaggt
      901 agcatgaacg caaacgacaa cgtggtgatt gttggcactg gtctggctgg cgttgaagtt
      961 gcgtttggtc tgcgtgcaag cggctgggaa ggtaacatcc gcctggtggg tgatgcgact
     1021 gttattccgc accacctgcc gccgctgtct aaagcgtacc tggcgggcaa ggcgactgcg
     1081 gagtctctgt acctgcgcac tccggatgct tacgcggctc agaacatcca gctgctgggc
     1141 ggtacccagg ttaccgcgat taaccgtgac cgtcagcagg tgattctgtc tgatggccgt
     1201 gcactggatt atgatcgtct ggtgctggca actggcggtc gtccgcgtcc gctgccggtg
     1261 gctagcggcg ctgttggtaa agcaaacaac ttccgttatc tgcgtaccct ggaagacgca
     1321 gaatgcatcc gtcgccagct gattgcggac aaccgtctgg ttgttatcgg tggcggctac
     1381 atcggtctgg aggttgctgc tactgcgatc aaagcgaaca tgcacgtgac tctgctggac
     1441 actgctgcgc gtgttctgga acgtgtgact gctccgccgg tgagcgcatt ctacgagcat
     1501 ctgcaccgcg aagctggtgt ggacattcgt actggtactc aggtttgcgg ctttgaaatg
     1561 tctaccgatc agcagaaagt tactgcggtt ctgtgcgaag acggcactcg tctgccggct
     1621 gatctggtta ttgcgggtat tggcctgatc ccgaactgcg aactggcatc cgcagcgggc
     1681 ctgcaggttg acaacggcat cgtgatcaac gagcacatgc agactagcga tccgctgatt
     1741 atggcagtgg gcgattgtgc gcgtttccac tctcagctgt atgaccgttg ggttcgcatc
     1801 gaaagcgtgc cgaacgcgct ggaacaggcg cgtaaaatcg cagcaatcct gtgcggtaaa
     1861 gttccgcgtg acgaggcggc tccgtggttt tggagcgacc agtatgaaat cggcctgaaa
     1921 atggtgggcc tgtccgaagg ttacgaccgt atcatcgttc gtggctctct ggcgcagccg
     1981 gacttctctg ttttctatct gcagggtgat cgcgttctgg cagttgacac tgtgaaccgt
     2041 ccggtggaat ttaaccagtc taaacagatc attactgatc gcctgccggt tgagccgaac
     2101 ctgctgggtg acgaatctgt gccgctgaag gagatcatcg cggctgcaaa agctgaactg
     2161 tcttctgcgt gaggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca
     2221 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2281 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     2341 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     2401 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2461 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2521 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2581 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2641 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2701 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2761 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     2821 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2881 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     2941 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3001 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3061 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3121 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3181 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     3241 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3301 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     3361 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     3421 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     3481 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     3541 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     3601 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     3661 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     3721 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     3781 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3841 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3901 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3961 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4021 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4081 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4141 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4201 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4261 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4321 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4381 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4441 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4501 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4561 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4621 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4681 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4741 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4801 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4861 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4921 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4981 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     5041 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     5101 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5161 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5221 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5281 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5341 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5401 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5461 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5521 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     5581 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5641 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     5701 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     5761 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     5821 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     5881 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5941 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     6001 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     6061 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     6121 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     6181 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     6241 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     6301 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     6361 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     6421 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     6481 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     6541 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     6601 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     6661 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     6721 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     6781 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     6841 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     6901 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     6961 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     7021 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     7081 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     7141 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     7201 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     7261 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     7321 ggcgtagagg atcgagatct cgatcccgcg aaat
//
