LOCUS       sequence-258706-        5001 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 5001)
  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 5001)
  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: pQE30-His-RF3
FEATURES             Location/Qualifiers
     source          1..5001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    30..46
                     /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             65..76
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             95..112
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             119..133
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     terminator      1748..1842
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1886..2545
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1952..1971)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2610..2696
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2743..2765)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2851..2991
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2906..2925
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3006..3023)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3177..3765)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3257..3276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3936..4727,4728..4796))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4559..4578
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4797..4901)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4969..4987
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaatttc acacagaatt
       61 cattaaagag gagaaattaa ctatgagagg atcgcatcac catcaccatc acggatccga
      121 tgacgatgac aaaatgacgt tgtctcctta tttgcaagag gtggcgaagc gccgcacttt
      181 tgccattatt tctcacccgg acgccggtaa gactaccatc accgagaagg tgctgctgtt
      241 cggacaggcc attcagaccg ccggtacagt aaaaggccgt ggttccaacc agcacgctaa
      301 gtcggactgg atggagatgg aaaagcagcg tgggatctcc attactacgt ctgtgatgca
      361 gtttccgtat cacgattgcc tggttaacct gctcgacacc ccggggcacg aagacttctc
      421 ggaagatacc tatcgtaccc tgacggcggt ggactgctgc ctgatggtta tcgacgccgc
      481 aaaaggtgtt gaagatcgta cccgtaagct gatggaagtt acccgtctgc gcgacacgcc
      541 gatcctcacc tttatgaaca aacttgaccg tgatatccgc gacccgatgg agctgctcga
      601 tgaagttgag aacgagctga aaatcggctg tgcgccgatc acctggccga ttggctgcgg
      661 caagctgttt aaaggcgttt accaccttta taaagacgaa acctatctct atcagagcgg
      721 taaaggccac accattcagg aagtccgcat tgttaaaggg ctgaataacc cggatctcga
      781 tgctgcggtt ggtgaagatc tggcacagca gctgcgtgac gaactggaac tggtgaaagg
      841 cgcgtctaac gagttcgaca aagagctgtt ccttgcgggc gaaatcactc cggtattctt
      901 cggtactgcg ctgggtaact tcggcgtcga tcatatgttg gatggcctgg tggagtgggc
      961 acctgcgccg atgccgcgtc agactgatac ccgtaccgta gaagcgagcg aagataaatt
     1021 taccggcttc gtatttaaaa ttcaggccaa catggacccg aaacaccgcg accgcgtggc
     1081 gtttatgcgt gtggtgtccg gtaaatatga aaaaggcatg aaactgcgcc aggtgcgcac
     1141 tgcgaaagat gtggtgatct ccgacgcgct gacctttatg gcgggtgacc gttcgcacgt
     1201 tgaagaagcg tatccgggcg atatcctcgg cctgcacaac cacggcacca ttcagatcgg
     1261 cgacaccttt acccagggtg agatgatgaa gttcaccggt attccgaact tcgcaccaga
     1321 actgttccgt cgtatccgcc tgaaagatcc gctgaagcaa aaacagctgc tcaaagggct
     1381 ggtacagctt tccgaagagg gcgcggtgca ggtgttccgt ccaatctcca acaacgatct
     1441 gatcgttggt gcagttggtg tgctgcagtt tgatgtggtg gtagcgcgcc tgaagagcga
     1501 atacaacgtt gaagcagtgt atgagtcagt caacgttgcc actgcccgct gggtagaatg
     1561 tgcagacgcg aagaaattcg aagagttcaa gcgtaagaac gaaagccaac tggcgcttga
     1621 tggcggcgat aacctcgctt acatcgctac cagcatggtc aacctgcgcc tggcacagga
     1681 acgttatccg gacgttcagt tccaccagac ccgcgagcat taattcaagc ttaattagct
     1741 gagcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     1801 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaagcta gcttggcgag
     1861 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     1921 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     1981 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     2041 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     2101 atttcgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     2161 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     2221 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     2281 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     2341 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     2401 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     2461 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     2521 cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc
     2581 tggggtaatg actctctagc ttgaggcatc aaataaaacg aaaggctcag tcgaaagact
     2641 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     2701 cctctagagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     2761 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     2821 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     2881 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     2941 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     3001 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3061 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3121 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3181 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3241 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3301 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3361 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3421 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3481 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3541 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3601 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3661 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3721 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3781 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3841 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3901 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3961 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4021 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4081 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4141 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4201 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4261 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4321 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4381 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4441 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4501 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4561 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4621 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4681 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4741 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4801 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4861 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4921 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4981 acgaggccct ttcgtcttca c
//
