LOCUS       sequence-258254-        6095 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 6095)
  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 6095)
  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-IPP1-His
FEATURES             Location/Qualifiers
     source          1..6095
                     /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             3441..3463
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3478..3502
                     /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(3502..3521)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3503..3521)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3609..3628)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3834..3911
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3912..4994
                     /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(3931..3950)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5803..5994
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5988..6010)
                     /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 agaacagaac
     2581 cggagatgaa gaaccacttg tcaatagact tgtcaattgg agcatctgcc tttggagaag
     2641 ctggtgggat ggcatcagag gcagccttgg agtaggttgg ggtgtcaggc aaagtaacat
     2701 tggtcaaatc aatacccttg ctgtcagaag acttaccagc aattaattgt ttccaggagt
     2761 catgtgtttc cttgatgata tccaaagcgt acttcttgtt cttagcttca ccggagaagg
     2821 caaattggtt ttctggctta ccatctggga ttttgtaaat tctgaaccat tcgttagtag
     2881 ccctcaacag acctgggaag tatttctcaa catcctcaat gtcgttcaat tttggggcta
     2941 atggatcgtt aatatcaatg gcaataactt tccaatcggt ctcaccttca tccaataaag
     3001 ccatgatacc tagagcctta acttgcttga cttgaccagt gtaagcaata gtttcaccaa
     3061 tttccaacac atcaattgga tcgttgtcac caactgcctt agtttctggg tggcttacgt
     3121 ttgggtcttc ccaagtttgt gggaaagcac catagttgtg aatgtaacca tggtgaggga
     3181 aacagtttct aacaaatctc aacttgccct tcttggtgtc ttggatgatt gggttcaaag
     3241 tttcttcctt ggtgatttct aacttggcgt tggtccaacg tggaatttca acaaccatgt
     3301 tgaaaatgtt gttttccttg tcagcgtaca agggaatgtc gtggaaggca gaaactggct
     3361 taccatcctt ttcgatataa actttgtatt ccaaggtgtt cttggcacca atttgtctgg
     3421 tagtgtaggt catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga
     3481 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
     3541 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     3601 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
     3661 catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
     3721 cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
     3781 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca
     3841 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     3901 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     3961 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     4021 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     4081 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     4141 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     4201 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     4261 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     4321 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     4381 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     4441 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     4501 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     4561 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     4621 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     4681 ttaccgagtc cgggctgcgc gttggtgcgg acatctcggt agtgggatac gacgataccg
     4741 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     4801 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     4861 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     4921 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4981 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga
     5041 tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     5101 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     5161 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
     5221 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
     5281 cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg
     5341 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     5401 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     5461 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     5521 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     5581 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     5641 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     5701 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     5761 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     5821 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     5881 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     5941 gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     6001 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     6061 cagggcgcgt cagcgggtgt tggcgggtgt cgggg
//
