LOCUS       sequence-208652-        6117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. solfataricus PCNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6117)
  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 6117)
  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: pS3GG
FEATURES             Location/Qualifiers
     source          1..6117
                     /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             982..999
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1052..1070)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1066..1113
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1150..1605
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1237..1256)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1447..1468
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1697..2512)
                     /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(1697..2512)
                     /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     2420..2439
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2504..2523
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2634..3222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3123..3142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3376..3393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3408..3547
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3473..3492)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3633..3655
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3649..3840)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4649..5731)
                     /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     5693..5712
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5732..5809)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6015..6034
                     /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 atctacctga aatctttcga acgtaacatc
      181 cgcctgatca atatgaaagt ggtttacgat gatgttcgtg tgctgaaaga tattatccag
      241 gcgctggccc gcctggttga tgaagccgtg ctgaaattca aacaggatag cgttgaactg
      301 gtggcactgg atcgtgcgca tattagcctg atctctgtta acctgccgcg cgaaatgttc
      361 aaagaatacg atgtgaacga tgaatttaaa ttcggtttca acacccagta cctgatgaaa
      421 atcctgaaag ttgcaaaacg taaagaagcc attgaaatcg caagtgaaag cccggatagt
      481 gtgattatca acattatcgg cagcaccaat cgtgaattta acgttcgcaa tctggaagtg
      541 tctgaacagg aaattccgga aatcaacctg cagttcgata ttagtgcgac gatcagctct
      601 gatggtttta aaagtgccat cagcgaagtt tctaccgtga cggataatgt ggttgtggaa
      661 ggtcacgaag atcgcattct gatcaaagca gaaggcgaaa gcgaagttga agtggaattt
      721 tctaaagata ccggcggtct gcaggatctg gaatttagta aagaaagcaa aaattcttac
      781 agtgcggaat acctggatga tgttctgtct ctgacgaaac tgagtgatta cgtgaaaatc
      841 agcttcggca accagaaacc gctgcagctg tttttcaata tggaaggcgg tggcaaagtt
      901 acgtacctgc tggcgccgaa agtgggagtg agaccggatc cgaattcgag ctccgtcgac
      961 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     1021 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1081 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1141 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1201 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1261 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1321 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1381 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1441 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1501 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1561 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     1621 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1681 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     1741 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1801 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1861 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     1921 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     1981 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2041 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2101 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2161 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2221 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2281 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2341 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2401 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2461 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     2521 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     2581 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2641 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2701 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2761 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2821 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2881 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2941 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3001 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3061 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3421 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     3481 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     3541 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3601 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3661 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     3721 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     3781 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     3841 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     3901 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     3961 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4021 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4081 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4141 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4201 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4261 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     4321 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     4381 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     4441 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     4501 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     4561 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     4621 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4681 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4741 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     4801 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     4861 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     4921 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4981 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     5041 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     5101 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     5161 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     5221 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     5281 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     5341 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     5401 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     5461 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     5521 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     5581 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     5641 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     5701 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     5761 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     5821 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     5881 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     5941 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     6001 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     6061 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
