LOCUS       sequence-236272-        4826 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 4826)
  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 4826)
  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-AsnRS
FEATURES             Location/Qualifiers
     source          1..4826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1573..1667
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1711..2370
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1777..1796)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2435..2521
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2568..2590)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2676..2816
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2731..2750
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2831..2848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3002..3590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3082..3101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3761..4552,4553..4621))
                     /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     4384..4403
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4622..4726)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4794..4812
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc atgagcgttg tgcctgtagc cgacgtactc
      181 cagggccgtg tagccgttga cagcgaagtc accgtgcgcg gatgggtacg tacccgccga
      241 gattcaaaag ctggcatctc cttcctcgcc gtttatgacg gttcctgctt tgatcctgta
      301 caggctgtca tcaataattc tctgcccaat tacaatgaag acgtcctgcg tctgaccacc
      361 ggctgctcgg tcattgtgac gggtaaagtc gtggcgtcgc cgggccaggg gcaacaattt
      421 gaaattcagg ccagcaaggt tgaagttgct ggttgggttg aagatccaga cacttacccg
      481 atggcggcaa aacgccacag cattgagtat ctgcgtgaag tcgctcacct gcgtccgcgc
      541 acaaacctga ttggtgccgt cgcgcgcgtt cgccatacgc tggcgcaggc gctgcatcgc
      601 ttctttaacg agcagggatt cttctgggtt tcaacgccac tgattaccgc atctgatacc
      661 gaaggtgcag gcgaaatgtt ccgcgtttct acgctggatc tggaaaacct gccgcgtaac
      721 gatcagggca aagtggattt cgacaaagac ttctttggta aagagtcttt cctgaccgta
      781 tctggccagt tgaacggcga aacctacgct tgcgcattgt ccaaaattta taccttcggc
      841 ccgactttcc gtgctgaaaa ctccaacacc agccgtcacc tggcggaatt ctggatgctg
      901 gagccggaag tggcgtttgc taacctgaac gatattgcgg gtctggctga agccatgctg
      961 aaatatgtct tcaaagcggt tctcgaagaa cgcgctgacg acatgaaatt cttcgctgaa
     1021 cgcgtagata aagatgccgt ttcacgtctg gaacgcttca ttgaagccga ttttgcgcag
     1081 gtggattata ccgacgcagt gaccattctc gaaaactgcg gcaggaagtt tgaaaacccg
     1141 gtttactggg gagtcgatct ctcttctgag catgagcgtt atctggcgga agaacacttt
     1201 aaagcaccgg tagtggttaa aaactatccg aaagatatta aagcgttcta tatgcgcctt
     1261 aacgaagacg gtaaaaccgt tgcggctatg gacgttctgg ctccgggcat cggtgagatc
     1321 attggtggct cccagcgtga agaacgtctg gacgtgctgg acgagcgtat gctggaaatg
     1381 ggcctgaata aagaagatta ctggtggtat cgcgatctgc gtcgctacgg tactgttccg
     1441 cattcaggtt tcggtcttgg ttttgaacgt ctgattgctt acgtaactgg cgtgcaaaac
     1501 gtacgtgatg tgattccgtt cccacgtact ccgcgtaacg ccagcttcta aaagcttaat
     1561 tagctgagct tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     1621 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca agctagcttg
     1681 gcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
     1741 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
     1801 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
     1861 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     1921 ccggaatttc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct
     1981 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac
     2041 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac
     2101 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg
     2161 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt
     2221 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     2281 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     2341 tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa
     2401 acgcctgggg taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa
     2461 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     2521 tccgccctct agagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     2581 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     2641 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     2701 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     2761 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     2821 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2881 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2941 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3001 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3061 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3121 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3181 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3241 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3301 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3361 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3421 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     3481 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3541 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3601 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3661 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3721 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3781 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3841 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3901 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3961 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4021 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4081 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4141 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4201 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4261 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4321 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4381 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4441 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4501 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4561 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4621 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4681 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4741 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4801 gtatcacgag gccctttcgt cttcac
//
