LOCUS       sequence-258253-        5816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for PURE system components.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5816)
  AUTHORS   Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, 
            Ueda T
  TITLE     Cell-free translation reconstituted with purified components.
  JOURNAL   Nat Biotechnol. 2001 Aug;19(8):751-5. doi: 10.1038/90802.
  PUBMED    11479568
REFERENCE   2  (bases 1 to 5816)
  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"; doi: "10"; journalName: 
            "Nat Biotechnol"; date: "2001-08"; volume: "19"; issue: "8"; pages: 
            "751-5"
COMMENT     Sequence Label: pET29b-AK1-His
FEATURES             Location/Qualifiers
     source          1..5816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..140
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     56..75
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(155..172)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(326..914)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(406..425)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1025..1044)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1036..1851
                     /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             1036..1851
                     /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     complement(1109..1128)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1943..2398)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2080..2101)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2292..2311
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2435..2482)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2478..2496
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2549..2566)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3162..3184
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3199..3223
                     /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)."
     primer_bind     complement(3223..3242)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3224..3242)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3330..3349)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3555..3632
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3633..4715
                     /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     complement(3652..3671)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5524..5715
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5709..5731)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
       61 tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa
      121 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      181 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      241 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      301 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      361 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      421 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      481 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      541 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      601 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      661 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      721 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
      781 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      841 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      901 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      961 aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt
     1021 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta
     1081 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     1141 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     1201 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     1261 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     1321 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     1381 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     1441 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     1501 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     1561 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     1621 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     1681 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     1741 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     1801 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt
     1861 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     1921 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     1981 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2041 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2101 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2161 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2221 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2281 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2341 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     2401 cattcgccaa tccggatata gttcctcctt tcagcaaaaa acccctcaag acccgtttag
     2461 aggccccaag gggttatgct agttattgct cagcggtggc agcagccaac tcagcttcct
     2521 ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg gtggtgctcg agcagcttgt
     2581 cgaggtagga gcagacctgc tggaaaacct catccacggt gccctcggca ttcagctggc
     2641 ggacgatgcc ccggcccttg tagaaggcga tgacgggttc ggtagccttg tagtacgtct
     2701 ccaagcgctt cttgatggtc tcctcgttgt cgtccacccg cccgctggtc tctccccgct
     2761 tcagcagccg cttcaccatc gtctccttcc ccgcatccac gtagagcagc agcgtcgggg
     2821 gggcaatctt cttttcaaac tcctctccct gcttcacctc gcgagggtag ccgtcaatga
     2881 ggaaaccctt ggaggtgtct gccttagcca acatggcgtc ccgcagcatg tccagcaccg
     2941 tgtccagggg aaccagctcg cccttctcca tgatggcttg cagcttcttg ccccgctctg
     3001 agcccgagct gacctctgcc cggagcaggt ccccagtgga gaggtgagtg tacccatact
     3061 tgtgcacaat cttctcgcat tgcgtcccct tccctgagcc ggggccaccc accacaaaga
     3121 tgatcttgtg gtgcttcagt ttttctgtcg acatatgtat atctccttct taaagttaaa
     3181 caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat
     3241 taatttcgcg ggatcgagat cgatctcgat cctctacgcc ggacgcatcg tggccggcat
     3301 caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga
     3361 tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc
     3421 cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt
     3481 gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga
     3541 gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc
     3601 gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
     3661 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg
     3721 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca
     3781 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca
     3841 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac
     3901 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg
     3961 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
     4021 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg
     4081 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg
     4141 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa
     4201 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa
     4261 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca
     4321 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
     4381 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gacatctcgg
     4441 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca
     4501 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg
     4561 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     4621 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     4681 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag
     4741 ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc
     4801 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc
     4861 atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt
     4921 cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc
     4981 gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc
     5041 gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     5101 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     5161 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     5221 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     5281 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     5341 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     5401 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     5461 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     5521 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     5581 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     5641 tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac
     5701 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     5761 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcgggg
//
