LOCUS       sequence-258586-        4569 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 4569)
  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 4569)
  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-CKM
FEATURES             Location/Qualifiers
     source          1..4569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(576..1367,1368..1436))
                     /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     1199..1218
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1437..1541)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1609..1627
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1651..1665,1666..1671,1672..1688,1689..1695)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1671..1687
                     /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    1703..1719
                     /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             1738..1749
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1768..1785
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2957..3051
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3095..3754
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3161..3180)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3819..3905
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3952..3974)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4060..4200
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4115..4134
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4215..4232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4386..4569,1..405))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4466..4485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
       61 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      121 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      181 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      241 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      301 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      361 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
      421 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      481 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      541 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      601 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
      661 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
      721 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
      781 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
      841 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
      901 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      961 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1021 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1081 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1141 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1201 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1261 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1321 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1381 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1441 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1501 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1561 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     1621 acgaggccct ttcgtcttca cctcgagaaa tcataaaaaa tttatttgct ttgtgagcgg
     1681 ataacaatta taatagattc aattgtgagc ggataacaat ttcacacaga attcattaaa
     1741 gaggagaaat taactatgag aggatcgcat caccatcacc atcacggatc catgccgttc
     1801 agcagcaccc acaacaaaca caagctgaag ttctcagccg aggaggaatt cccggacctc
     1861 tcgaagcaca acaaccacat ggccaaagtc ctcaccccgg agctctacaa acgcctccgg
     1921 gataaggaga ccccgagcgg attcaccctc gacgacgtca tccaaaccgg ggtcgataac
     1981 cccggtcacc cattcatcat gacggtgggc tgcgtggcgg gggacgagga gtcctacgag
     2041 gtgttcaagg atctcttcga ccccgtcatc caggaccgcc acggggggta caaacccacc
     2101 gataagcacc gcaccgacct caaccacgag aacctcaagg ggggtgacga cctggacccc
     2161 aaatacgtgc tgagcagccg cgtgcgcacg gggagaagca ttaaggggta ctccctgccc
     2221 ccacactgca gccgtgggga gcggcgcgcc gtcgagaagc tgtccgtgga agccctgaac
     2281 agcctggagg gggagttcaa gggccgctat taccctctga aggccatgac ggagcaggag
     2341 cagcagcagc tgatcgacga ccacttcctg ttcgataaac ccgtctcccc actgctgctc
     2401 gcatccggga tggcccgaga ttggcccgac gccaggggca tctggcacaa cgacaacaag
     2461 acgttcctgg tgtgggtgaa tgaggaggac cacctgaggg tcatctccat ggagaaggga
     2521 ggaaatatga aggaggtctt ccggcgcttc tgcgtcggcc tcaagaagat cgaggagatc
     2581 ttcaagaagg ccgggcaccc cttcatgtgg acggagcacc tgggttacat cctgacgtgc
     2641 ccctccaatt tgggcacggg gctgcggggg ggggtccacg tgaagctccc caaactcagc
     2701 cagcacccca aattcgagga gatcctccat aggctgcgcc tgcagaaacg gggcaccggc
     2761 ggggtggata cggcggcggt gggcgccgtc tttgacatct ccaacgccga ccggctgggc
     2821 ttctcggagg tggagcaggt gcagatggtg gtggacggcg tcaagctcat ggtggagatg
     2881 gagaagaagc tggagcagaa ccagcccata gacgacatga tcccggccca gaagtaagct
     2941 taattagctg agcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
     3001 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagctag
     3061 cttggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     3121 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     3181 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     3241 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     3301 tcatccggaa tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     3361 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     3421 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     3481 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     3541 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     3601 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     3661 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     3721 acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc
     3781 ttaaacgcct ggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt
     3841 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     3901 caaatccgcc ctctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     3961 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     4021 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     4081 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4141 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4201 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4261 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4321 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4381 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4441 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4501 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4561 ggaagcgtg
//
