LOCUS       sequence-258530-        7828 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 7828)
  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 7828)
  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: pET16b-His-ArgRS
FEATURES             Location/Qualifiers
     source          1..7828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1281..2363)
                     /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     2325..2344
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2364..2441)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2647..2666
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2750..2769
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2750..2768
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2769..2793
                     /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             2808..2830
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2843..2872
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2885..2896
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5074..5092)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5088..5135
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5369..5387)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5455..5559
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5560..5628,5629..6420)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5778..5797)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6591..7179
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7080..7099
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7333..7350
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7365..7504
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7430..7449)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7590..7612
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7606..7797)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
       61 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      121 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      181 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      241 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      301 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      361 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      421 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
      481 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
      541 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
      601 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
      661 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
      721 cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga
      781 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
      841 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
      901 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
      961 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     1021 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     1081 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     1141 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     1201 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     1261 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1321 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     1381 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     1441 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     1501 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     1561 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     1621 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     1681 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     1741 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     1801 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     1861 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     1921 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     1981 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     2041 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     2101 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     2161 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     2221 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     2281 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     2341 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     2401 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     2461 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     2521 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     2581 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     2641 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     2701 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     2761 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     2821 aagaaggaga tataccatgg gccatcatca tcatcatcat catcatcatc acagcagcgg
     2881 ccatatcgaa ggtcgtcata tgaatattca ggctcttctc tcagaaaaag tccgtcaggc
     2941 catgattgcg gcaggcgcgc ctgcggattg cgaaccgcag gttcgtcagt cagcaaaagt
     3001 tcagttcggc gactatcagg ctaacggcat gatggcagtt gctaaaaaac tgggtatggc
     3061 accgcgacaa ttagcagagc aggtgctgac tcatctggat cttaacggta tcgccagcaa
     3121 agttgagatc gccggtccag gctttatcaa cattttcctt gatccggcat tcctggctga
     3181 acatgttcag caggcgctgg cgtccgatcg tctcggtgtt gctacgccag aaaaacagac
     3241 cattgtggtt gactactctg cgccaaacgt ggcgaaagag atgcatgtcg gtcacctgcg
     3301 ctctaccatt attggtgacg cagcagtgcg tactctggag ttcctcggtc acaaagtgat
     3361 tcgcgcaaac cacgtcggcg actggggcac tcagttcggt atgctgattg catggctgga
     3421 aaagcagcag caggaaaacg ccggtgaaat ggagctggct gaccttgaag gtttctaccg
     3481 cgatgcgaaa aagcattacg atgaagatga agagttcgcc gagcgcgcac gtaactacgt
     3541 ggtaaaactg caaagcggtg acgaatattt ccgcgagatg tggcgcaaac tggtcgacat
     3601 caccatgacg cagaaccaga tcacctacga tcgtctcaac gtgacgctga cccgtgatga
     3661 cgtgatgggc gaaagcctct acaacccgat gctgccagga attgtggcgg atctcaaagc
     3721 caaaggtctg gcagtagaaa gcgaaggggc gaccgtcgta ttccttgatg agtttaaaaa
     3781 caaggaaggc gaaccgatgg gcgtgatcat tcagaagaaa gatggcggct atctctacac
     3841 caccactgat atcgcctgtg cgaaatatcg ttatgaaaca ctgcatgccg atcgcgtgct
     3901 gtattacatc gactcccgtc agcatcaaca cctgatgcag gcatgggcga tcgtccgtaa
     3961 agcaggctat gtaccggaat ccgtaccgct ggaacaccac atgttcggca tgatgctggg
     4021 taaagacggc aaaccgttca aaacccgcgc gggtggtaca gtgaaactgg ccgatctgct
     4081 ggatgaagcc ctggaacgtg cacgccgtct ggtggcagaa aagaacccgg atatgccagc
     4141 cgacgagctg gaaaaactgg ctaacgcggt tggtattggt gcggtgaaat atgcggatct
     4201 ctccaaaaac cgcaccacgg actacatctt cgactgggac aacatgctgg cgtttgaggg
     4261 taataccgcg ccatacatgc agtatgcata cacgcgtgta ttgtccgtgt tccgtaaagc
     4321 agaaattgac gaagagcaac tggctgcagc tccggttatc atccgtgaag atcgtgaagc
     4381 gcaactggca gctcgcctgc tgcagtttga agaaaccctc accgtggttg cccgtgaagg
     4441 cacgccgcat gtaatgtgtg cttacctgta cgatctggcc ggtctgttct ctggcttcta
     4501 cgagcactgc ccgatcctca gcgcagaaaa cgaagaagtg cgtaacagcc gtctaaaact
     4561 ggcacaactg acggcgaaga cgctgaagct gggtctggat acgctgggta ttgagactgt
     4621 agagcgtatg taatcgattt ttcgtgagag tgaagcctga tcagggttag ccgatcaggc
     4681 ttttttattg ccatctaaat gtattctgaa aatggacatg ccattgtttt ctcactgttg
     4741 gataagaggc cagaagcgta atatccggcc ccagggaaac gataacggtt gaatttaagg
     4801 aataccgcag tgtttaaatt tcttgtatta acattaggca ttatctcttg ccaggcttac
     4861 gcagaagata cggttatagt aaacgaccat gacatttcag ccatcaaaga ttgttggcaa
     4921 aaaaattcag atgatgatac tgacgttaac gtgatcaaat catgcctgcg acaagaatac
     4981 aatctcgtcg atgcgcaatt aaataaagcc tatggtgaag cttggatccg gctgctaaca
     5041 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     5101 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     5161 atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt
     5221 gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg
     5281 ttagcaattt aactgtgata aactaccgca ttaaagctag cttatcgatg ataagctgtc
     5341 aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5401 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5461 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5521 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5581 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5641 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5701 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5761 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     5821 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5881 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5941 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6001 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6061 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     6121 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6181 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6241 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6301 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6361 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6421 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6481 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6541 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6601 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6661 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6721 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     6781 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6841 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6901 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6961 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7021 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7081 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7141 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7201 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7261 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     7321 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     7381 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     7441 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     7501 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     7561 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     7621 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     7681 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     7741 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     7801 tgcctccgtg taagggggat ttctgttc
//
