LOCUS       sequence-208599-        6075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M. sedula PCNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6075)
  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 6075)
  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: pM3GG
FEATURES             Location/Qualifiers
     source          1..6075
                     /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             940..957
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1010..1028)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1024..1071
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1108..1563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1195..1214)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1405..1426
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1655..2470)
                     /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(1655..2470)
                     /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     2378..2397
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2462..2481
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2592..3180
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3081..3100
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3334..3351
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3366..3505
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3431..3450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3591..3613
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3607..3798)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4607..5689)
                     /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     5651..5670
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5690..5767)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5973..5992
                     /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 cgtatcgcat acgcgaacgc gatggacttc
      181 aaaaccgtga tcgaagcgct gtctaaactg atcgacgaag tgaccttcac tttcaccagc
      241 tctggtctgg acgttgttgc ggttgaccgt gcgcatatca gcctgatcaa gctgcacttc
      301 ccgaaagaag cgttcgaaga atttgacgtg gaagaccagt tccgtttcgg ctttaacacc
      361 cagtacatgc tgaaggttat ggcgtctgcg aaacgcaagg agaagattga gatggagtcc
      421 cgtgaagagt ccgaaatcgt tatccgtatg ctgggcgaac cgccgcgtga atttaccatc
      481 cgtaacattg aggttccgat ccaggaactg ccggagctga aactggattt cgacgtgaag
      541 gcgaaaatca cttctggcgg cttcaagaag gctgttagcg agattgcgac cgtttctgac
      601 tctgtggaga ttgacgcgac cgaaatggag attaagctgc gttccaagga gtccaccgag
      661 atcgaggtgg agttctccaa agaaatgggt ggtctgcagg aaattgaggt gaagaagccg
      721 tctgtttctt cctacccgtc tgactacctg gaggacgttc tggttctgac ccgcctgtct
      781 ggcttcctga acctgctgtt ctctgagcag aaaccgctgc agctggagtt caacatggac
      841 aacggtggct ccgtggttta tctgctggcg ccgaacgttg gtggagtgag accggatccg
      901 aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga
      961 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1021 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1081 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1141 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1201 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1261 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1321 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1381 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1441 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1501 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1561 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1621 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     1681 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     1741 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     1801 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     1861 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     1921 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     1981 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     2041 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     2101 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     2161 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     2221 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     2281 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     2341 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     2401 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     2461 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     2521 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2581 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2641 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2701 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     2761 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2821 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2881 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2941 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3001 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3061 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3121 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3181 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3241 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3301 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3361 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3421 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3481 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     3541 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3601 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     3661 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     3721 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     3781 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     3841 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     3901 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3961 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     4021 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     4081 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     4141 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     4201 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     4261 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     4321 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     4381 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4441 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     4501 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4561 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4621 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4681 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     4741 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     4801 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     4861 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     4921 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4981 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     5041 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     5101 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     5161 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     5221 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5281 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5341 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5401 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     5461 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     5521 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     5581 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     5641 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     5701 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     5761 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     5821 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     5881 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     5941 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     6001 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     6061 tcgatcccgc gaaat
//
