LOCUS       sequence-257379-        5366 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 5366)
  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 5366)
  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-ThrRS
FEATURES             Location/Qualifiers
     source          1..5366
                     /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             151..165
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     terminator      2113..2207
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2251..2910
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2317..2336)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2975..3061
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3108..3130)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3216..3356
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3271..3290
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3371..3388)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3542..4130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3622..3641)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4301..5092,5093..5161))
                     /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     4924..4943
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5162..5266)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5334..5352
                     /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 gatgacgatg acaaacctgt tataactctt
      181 cctgatggca gccaacgcca ttacgatcac gctgtaagcc ccatggatgt tgcgctggac
      241 attggtccag gtctggcgaa agcctgtatc gcagggcgcg ttaatggcga actggttgat
      301 gcttgcgatc tgattgaaaa cgacgcacaa ctgtcgatca ttaccgccaa agacgaagaa
      361 ggtctggaga tcattcgtca ctcctgtgcg cacctgttag ggcacgcgat taaacaactt
      421 tggccgcata ccaaaatggc aatcggcccg gttattgaca acggttttta ttacgacgtt
      481 gatcttgacc gcacgttaac ccaggaagat gtcgaagcac tcgagaagcg gatgcatgag
      541 cttgctgaga aaaactacga cgtcattaag aagaaagtca gctggcacga agcgcgtgaa
      601 actttcgcca accgtgggga gagctacaaa gtctccattc ttgacgaaaa catcgcccat
      661 gatgacaagc caggtctgta cttccatgaa gaatatgtcg atatgtgccg cggtccgcac
      721 gtaccgaaca tgcgtttctg ccatcatttc aaactaatga aaacggcagg ggcttactgg
      781 cgtggcgaca gcaacaacaa aatgttgcaa cgtatttacg gtacggcgtg ggcagacaaa
      841 aaagcactta acgcttacct gcagcgcctg gaagaagccg cgaaacgcga ccaccgtaaa
      901 atcggtaaac agctcgacct gtaccatatg caggaagaag cgccgggtat ggtattctgg
      961 cacaacgacg gctggaccat cttccgtgaa ctggaagtgt ttgttcgttc taaactgaaa
     1021 gagtaccagt atcaggaagt taaaggtccg ttcatgatgg accgtgtcct gtgggaaaaa
     1081 accggtcact gggacaacta caaagatgca atgttcacca catcttctga gaaccgtgaa
     1141 tactgcatta agccgatgaa ctgcccgggt cacgtacaaa ttttcaacca ggggctgaag
     1201 tcttatcgcg atctgccgct gcgtatggcc gagtttggta gctgccaccg taacgagccg
     1261 tcaggttcgc tgcatggcct gatgcgcgtg cgtggattta cccaggatga cgcgcatatc
     1321 ttctgtactg aagaacaaat tcgcgatgaa gttaacggat gtatccgttt agtctatgat
     1381 atgtacagca cttttggctt cgagaagatc gtcgtcaaac tctccactcg tcctgaaaaa
     1441 cgtattggca gcgacgaaat gtgggatcgt gctgaggcgg acctggcggt tgcgctggaa
     1501 gaaaacaaca tcccgtttga atatcaactg ggtgaaggcg ctttctacgg tccgaaaatt
     1561 gaatttaccc tgtatgactg cctcgatcgt gcatggcagt gcggtacagt acagctggac
     1621 ttctctttgc cgtctcgtct gagcgcttct tatgtaggcg aagacaatga acgtaaagta
     1681 ccggtaatga ttcaccgcgc aattctgggg tcgatggaac gtttcatcgg tatcctgacc
     1741 gaagagttcg ctggtttctt cccgacctgg cttgcgccgg ttcaggttgt tatcatgaat
     1801 attaccgatt cacagtctga atacgttaac gaattgacgc aaaaactatc aaatgcgggc
     1861 attcgtgtta aagcagactt gagaaatgag aagattggct ttaaaatccg cgagcacact
     1921 ttgcgtcgcg tcccatatat gctggtctgt ggtgataaag aggtggaatc aggcaaagtt
     1981 gccgttcgca cccgccgtgg taaagacctg ggaagcatgg acgtaaatga agtgatcgag
     2041 aagctgcaac aagagattcg cagccgcagt cttaaacaat tggaggaata aaagcttaat
     2101 tagctgagct tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     2161 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca agctagcttg
     2221 gcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
     2281 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
     2341 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
     2401 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     2461 ccggaatttc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct
     2521 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac
     2581 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac
     2641 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg
     2701 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt
     2761 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     2821 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     2881 tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa
     2941 acgcctgggg taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa
     3001 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     3061 tccgccctct agagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3121 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3181 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     3241 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     3301 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     3361 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3421 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3481 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3541 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3601 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3661 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3721 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3781 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3841 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3901 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3961 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4021 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4081 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4141 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4201 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4261 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4321 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4381 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4441 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4501 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4561 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4621 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4681 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4741 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4801 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4861 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4921 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4981 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5041 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5101 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5161 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5221 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5281 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5341 gtatcacgag gccctttcgt cttcac
//
