LOCUS       sequence-258527-        6074 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 6074)
  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 6074)
  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-AlaRS
FEATURES             Location/Qualifiers
     source          1..6074
                     /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"
     CDS             157..171
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     terminator      2821..2915
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2959..3618
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3025..3044)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3683..3769
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3816..3838)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3924..4064
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3979..3998
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4079..4096)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4250..4838)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4330..4349)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5009..5800,5801..5869))
                     /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     5632..5651
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5870..5974)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6042..6060
                     /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 gcatgcgatg acgatgacaa aagcaacagc
      181 accgctgaga tccgtcaggc gtttctcgac tttttccata gtaagggaca tcaggtagtt
      241 gccagcagct ccctggtacc ccataacgac ccaactttgt tgtttaccaa cgccgggatg
      301 aaccagttca aggatgtgtt ccttgggctc gacaagcgta attattcccg cgctaccact
      361 tcccaacgct gcgtgcgtgc gggtggtaaa cacaacgacc tggaaaacgt cggttacacc
      421 gcgcgtcacc ataccttctt cgaaatgctg ggcaacttca gcttcggcga ctatttcaaa
      481 cacgatgcca ttcagtttgc atgggaactg ctgaccagcg aaaaatggtt tgccctgccg
      541 aaagagcgtc tgtgggttac cgtctatgaa agcgacgacg aagcctacga aatctgggaa
      601 aaagaagtag ggatcccgcg cgaacgtatt attcgcatcg gcgataacaa aggtgcgcca
      661 tacgcatctg acaacttctg gcagatgggt gacactggtc cgtgcggccc gtgcaccgaa
      721 atcttctacg atcacggcga ccacatttgg gggggccctc cgggaagccc ggaagaagac
      781 ggcgaccgct acattgagat ctggaacatc gtcttcatgc agttcaaccg ccaggccgat
      841 ggcacgatgg aaccgctgcc gaagccgtct gtagataccg gtatgggtct ggagcgtatt
      901 gctgcggtgc tgcaacacgt taactctaac tatgacatcg acctgttccg cacgctgatc
      961 caggcggtag cgaaagtcac tggcgcaacc gatctgagca ataaatcgct gcgcgtaatc
     1021 gctgaccaca ttcgttcttg tgcgttccta atcgcggatg gcgtaatgcc gtccaatgaa
     1081 aaccgtggtt atgtactgcg tcgtatcatt cgtcgcgcag tgcgtcacgg taatatgctc
     1141 ggcgcgaaag aaaccttctt ctacaaactg gttggtccgc tgatcgacgt tatgggctct
     1201 gcgggtgaag acctgaaacg ccagcaggcg caggttgagc aggtgctgaa gactgaagaa
     1261 gagcagtttg ctcgtactct ggagcgcggt ctggcgttgc tggatgaaga gctggcaaaa
     1321 ctttctggtg atacgctgga tggtgaaact gctttccgtc tgtacgacac ctatggcttc
     1381 ccggttgacc tgacggctga tgtttgtcgt gagcgcaaca tcaaagttga cgaagctggt
     1441 tttgaagctg caatggaaga gcagcgtcgt cgcgcgcgcg aagccagcgg ctttggtgcc
     1501 gattacaacg caatgatccg tgttgacagt gcatctgaat ttaaaggcta tgaccatctg
     1561 gaactgaacg gcaaagtgac tgcgctgttt gttgatggta aagcggttga tgccatcaat
     1621 gcaggccagg aagctgtggt cgtgctggat caaacgccat tctatgcgga atccggcggt
     1681 caggttggcg ataaaggcga actgaaaggc gctaacttct cctttgcggt ggaagatacg
     1741 cagaaatacg gccaggcgat tggtcacatc ggtaaacttg ctgcgggttc tctgaaagtg
     1801 ggcgacgcgg tgcaggctga tgttgatgag gctcgtcgcg cccgtattcg tctgaatcac
     1861 tccgcaacgc acctgatgca cgctgcgctg cgccaggttc tgggtactca tgtatcgcag
     1921 aaaggttcac tggttaacga caaggtgctg cgcttcgact tctcacacaa cgaagcgatg
     1981 aaaccagaag agattcgtgc ggtcgaagac ctggtgaaca cacagattcg tcgcaatttg
     2041 ccgatcgaaa ccaacatcat ggatctcgaa gcggcgaaag cgaaaggtgc gatggcgctg
     2101 ttcggcgaga agtatgatga gcgcgtacgc gtgctgagca tgggcgattt ctctaccgag
     2161 ttgtgtggcg gtactcacgc cagccgcact ggtgatattg gtctgttccg catcatctct
     2221 gaatcgggta ctgctgcagg cgttcgtcgt atcgaagcgg taaccggaga aggtgctatc
     2281 gccaccgttc atgcagacag cgatcgctta agcgaagtcg cgcatctgct gaaaggcgat
     2341 agcaataatc tggctgataa agtgcgctca gtactggaac gtacgcgtca gctggaaaaa
     2401 gagttacaac agcttaaaga acaagctgcc gcacaggaga gcgcaaatct ttccagtaag
     2461 gcaattgatg ttaatggtgt taagctgttg gttagcgagc ttagcggtgt tgagccgaaa
     2521 atgttgcgta ccatggttga cgatttaaaa aatcagctgg ggtcgacaat tatcgtgctg
     2581 gcaacggtag tcgaaggtaa ggtttctctg attgcaggcg tatctaagga cgtcacagat
     2641 cgtgtgaaag caggggaact gattggtatg gtcgctcagc aggtgggcgg caagggtggt
     2701 ggacgtcctg acatggcgca agccggtggt acggatgctg cggccttacc tgcagcgtta
     2761 gccagtgtga aaggctgggt cagcgcgaaa ttgcaataaa agcttaatta gctgagcttg
     2821 gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc
     2881 tcggttgccg ccgggcgttt tttattggtg agaatccaag ctagcttggc gagattttca
     2941 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     3001 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     3061 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     3121 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaatttcgt
     3181 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     3241 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     3301 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     3361 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     3421 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     3481 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     3541 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     3601 tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggggta
     3661 atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3721 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccctctag
     3781 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3841 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3901 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     3961 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     4021 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     4081 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4141 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4201 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4261 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4321 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4381 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4441 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4501 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4561 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4621 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4681 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4741 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4801 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4861 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4921 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4981 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5041 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5101 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5161 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5221 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5281 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5341 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5401 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5461 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5521 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5581 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5641 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5701 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5761 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5821 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5881 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5941 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6001 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     6061 cctttcgtct tcac
//
