LOCUS       sequence-258692-        6829 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 6829)
  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 6829)
  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-PheRS
FEATURES             Location/Qualifiers
     source          1..6829
                     /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      3576..3670
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3714..4373
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3780..3799)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4438..4524
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4571..4593)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4679..4819
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4734..4753
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4834..4851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5005..5593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5085..5104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5764..6555,6556..6624))
                     /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     6387..6406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6625..6729)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6797..6815
                     /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 atcacatctc
      181 gcagaactgg ttgccagtgc gaaggcggcc attagccagg cgtcagatgt tgccgcgtta
      241 gataatgtgc gcgtcgaata tttgggtaaa aaagggcact taacccttca gatgacgacc
      301 ctgcgtgagc tgccgccaga agagcgtccg gcagctggtg cggttatcaa cgaagcgaaa
      361 gagcaggttc agcaggcgct gaatgcgcgt aaagcggaac tggaaagcgc tgcactgaat
      421 gcgcgtctgg cggcggaaac gattgatgtc tctctgccag gtcgtcgcat tgaaaacggc
      481 ggtctgcatc cggttacccg taccatcgac cgtatcgaaa gtttcttcgg tgagcttggc
      541 tttaccgtgg caaccgggcc ggaaatcgaa gacgattatc ataacttcga tgctctgaac
      601 attcctggtc accacccggc gcgcgctgac cacgacactt tctggtttga cactacccgc
      661 ctgctgcgta cccagacctc tggcgtacag atccgcacca tgaaagccca gcagccaccg
      721 attcgtatca tcgcgcctgg ccgtgtttat cgtaacgact acgaccagac tcacacgccg
      781 atgttccatc agatggaagg tctgattgtt gataccaaca tcagctttac caacctgaaa
      841 ggcacgctgc acgacttcct gcgtaacttc tttgaggaag atttgcagat tcgcttccgt
      901 ccttcctact tcccgtttac cgaaccttct gcagaagtgg acgtcatggg taaaaacggt
      961 aaatggctgg aagtgctggg ctgcgggatg gtgcatccga acgtgttgcg taacgttggc
     1021 atcgacccgg aagtttactc tggtttcgcc ttcgggatgg ggatggagcg tctgactatg
     1081 ttgcgttacg gcgtcaccga cctgcgttca ttcttcgaaa acgatctgcg tttcctcaaa
     1141 cagtttaaat aaggcaggaa tagattatga aattcggtga actgtggtta cgcgaatggg
     1201 tgaacccggc gattgatagc gatgcgctgg caaatcaaat cactatggcg ggcctggaag
     1261 ttgacggtgt agaaccggtt gccggcagct tccacggcgt ggtcgttggt gaagtggttg
     1321 agtgtgcgca gcatccgaac gctgacaaac tgcgtgtgac aaaagtgaat gtcggcggcg
     1381 atcgcctgct ggacatcgtc tgcggtgcgc caaactgccg tcagggcctg cgtgtagcgg
     1441 tagcgaccat tggtgctgtt ctgccgggtg atttcaaaat taaagcggcg aaactgcgtg
     1501 gcgaaccgtc tgaagggatg ctgtgctcct tctctgaact gggcatttct gacgatcaca
     1561 gcggcattat cgaactgcct gcggatgcgc cgattggcac cgatatccgt gaatacctga
     1621 aacttgatga caacaccatc gaaatcagcg tgacgccaaa ccgtgccgac tgcttaggca
     1681 tcattggtgt tgcgcgtgac gttgccgtgc tgaaccagct gccgctggtt caaccggaaa
     1741 tcgttccggt tggtgcgacc atcgacgaca cgctgccgat tacagtcgaa gcgccggaag
     1801 cctgcccgcg ttatcttggc cgtgtggtaa aaggcattaa cgttaaagcg ccaactccgc
     1861 tgtggatgaa agaaaaactg cgtcgttgcg ggatccgttc tatcgatgca gttgttgacg
     1921 tcaccaacta tgtgctgctc gaactgggcc agccgatgca cgctttcgat aaagatcgca
     1981 ttgaaggcgg cattgtggtg cggatggcga aagagggcga aacgctggtg ctgctcgacg
     2041 gtactgaagc gaagctgaat gctgacactc tggtcatcgc cgaccacaac aaggcgctgg
     2101 cgatgggcgg catcttcggt ggcgaacact ctggcgtgaa tgacgaaaca caaaacgtgc
     2161 tgctggaatg cgcgttcttt agcccgctgt ctatcaccgg tcgtgctcgt cgtcatggcc
     2221 tgcataccga tgcgtctcac cgttatgagc gtggcgttga tccggcactg cagcacaaag
     2281 cgatggaacg tgcgacccgt ctgctgatcg acatctgcgg tggtgaggct ggcccggtaa
     2341 ttgatatcac caacgaagca acgctgccga agcgtgcaac catcactcta cgtcgtagca
     2401 aactggatcg cctgatcggc catcatattg cggatgagca ggtaactgac attctgcgtc
     2461 gtctcggctg cgaagtgacc gaaggcaaag acgaatggca ggcagttgcg ccgagctggc
     2521 gtttcgatat ggagattgaa gaagatctgg ttgaagaagt cgcgcgtgtt tacggctaca
     2581 acaacatccc ggatgagccg gtacaggcaa gcctgattat gggtactcac cgtgaagctg
     2641 acctgtcgct caagcgcgtg aaaacgctgc tcaacgacaa aggctatcag gaagtgatca
     2701 cctacagctt cgttgatccg aaagtgcagc agatgatcca tccaggcgtt gaagccttac
     2761 tgctgccaag cccgatctct gttgaaatgt cagcaatgcg tctttctctg tggactggcc
     2821 tgctggcaac cgtggtgtac aaccagaacc gtcagcagaa ccgtgtgcgc attttcgaaa
     2881 gcggtctgcg tttcgtacca gatactcagg caccgttggg cattcgtcag gatctgatgt
     2941 tagccggtgt gatttgcggt aaccgttacg aagagcactg gaacctggca aaagagaccg
     3001 ttgatttcta tgatttgaaa ggcgatcttg aatccgttct cgacctgacc ggtaaactga
     3061 atgaggttga gttccgtgca gaagcgaatc cggcactgca tccggggcaa tccgcagcga
     3121 tttatctgaa aggtgaacgt attggttttg ttggggttgt tcatcctgaa ctggaacgta
     3181 aactggatct taacggtcgc actctggtgt tcgaactgga gtggaacaag ctcgcagacc
     3241 gcgtggtgcc tcaggcgcgc gagatttctc gcttcccggc gaaccgtcgt gacatcgcgg
     3301 tggtggtcgc agaaaacgtt cccgcagcgg atattttatc cgaatgtaag aaagttggcg
     3361 taaatcaggt agttggcgta aacttatttg acgtgtaccg cggtaagggt gttgcggagg
     3421 ggtataagag cctcgccata agcctgatcc tgcaagatac cagccgtaca ctcgaagaag
     3481 aggagattgc cgctaccgtc gccaaatgtg tagaggcatt aaaagagcga ttccaggcat
     3541 cattgaggga ttgaaagctt aattagctga gcttggactc ctgttgatag atccagtaat
     3601 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
     3661 tggtgagaat ccaagctagc ttggcgagat tttcaggagc taaggaagct aaaatggaga
     3721 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     3781 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     3841 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     3901 ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac ggtgagctgg
     3961 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     4021 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     4081 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     4141 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     4201 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     4261 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     4321 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taattttttt
     4381 aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt gaggcatcaa
     4441 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     4501 aacgctctcc tgagtaggac aaatccgccc tctagagctg cctcgcgcgt ttcggtgatg
     4561 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4621 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4681 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     4741 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     4801 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4861 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4921 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4981 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5041 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5101 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5161 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5221 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5281 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5341 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5401 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5461 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5521 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5581 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5641 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5701 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5761 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5821 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5881 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5941 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6001 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6061 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6121 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6181 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6241 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6301 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6361 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6421 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6481 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6541 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6601 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6661 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6721 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     6781 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcac
//
