LOCUS       sequence-208648-        6081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M. sedula PCNA1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6081)
  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 6081)
  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: pM1GG
FEATURES             Location/Qualifiers
     source          1..6081
                     /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             946..963
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1016..1034)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1030..1077
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1114..1569
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1201..1220)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1411..1432
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1661..2476)
                     /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(1661..2476)
                     /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     2384..2403
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2468..2487
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2598..3186
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3087..3106
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3340..3357
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3372..3511
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3437..3456)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3597..3619
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3613..3804)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4613..5695)
                     /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     5657..5676
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5696..5773)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5979..5998
                     /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 agtgagaccg
      901 gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
      961 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1021 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1081 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1141 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1201 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1261 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1321 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1381 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1441 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1501 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1561 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1621 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1681 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1741 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1801 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1861 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1921 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1981 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     2041 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     2101 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     2161 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2221 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2281 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2341 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2401 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2461 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2521 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2581 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2641 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2701 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2761 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2821 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2881 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2941 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3001 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3061 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3121 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3181 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3241 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3301 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3361 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3421 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3481 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3541 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3601 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3661 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3721 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3781 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3841 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     3901 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     3961 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4021 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4081 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4141 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4201 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4261 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4321 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4381 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4441 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4501 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4561 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4621 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4681 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4741 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4801 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4861 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4921 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4981 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5041 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5101 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5161 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5221 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5281 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5341 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5401 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5461 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5521 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5581 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5641 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5701 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5761 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5821 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5881 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     5941 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6001 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     6061 gagatctcga tcccgcgaaa t
//
