LOCUS       sequence-258588-        3883 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 3883)
  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 3883)
  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-NDK
FEATURES             Location/Qualifiers
     source          1..3883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(532..1323,1324..1392))
                     /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     1155..1174
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1393..1497)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1565..1583
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1607..1621,1622..1627,1628..1644,1645..1651)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1627..1643
                     /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    1659..1675
                     /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             1694..1705
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1724..1741
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1748..1762
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     terminator      2227..2321
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2365..3024
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2431..2450)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3089..3175
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3222..3244)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3330..3470
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3385..3404
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3485..3502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3656..3883,1..361))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3736..3755)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
       61 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
      121 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
      181 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
      241 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
      301 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
      361 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
      421 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
      481 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
      541 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
      601 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
      661 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
      721 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
      781 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
      841 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
      901 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
      961 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     1021 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     1081 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     1141 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     1201 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     1261 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     1321 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     1381 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     1441 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     1501 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     1561 taaaaatagg cgtatcacga ggccctttcg tcttcacctc gagaaatcat aaaaaattta
     1621 tttgctttgt gagcggataa caattataat agattcaatt gtgagcggat aacaatttca
     1681 cacagaattc attaaagagg agaaattaac tatgagagga tcgcatcacc atcaccatca
     1741 cggatccgat gacgatgaca aagctattga acgtactttt tccatcatca aaccgaacgc
     1801 ggtagcaaaa aacgtcattg gtaatatctt tgcgcgcttt gaagctgcag ggttcaaaat
     1861 tgttggcacc aaaatgctgc acctgaccgt tgaacaggca cgtggctttt atgctgaaca
     1921 cgatggaaaa ccgttctttg atggtctggt tgaattcatg acctctggcc cgatcgtggt
     1981 ttccgtgctg gaaggtgaaa acgccgttca gcgtcaccgc gatctgctgg gcgcgaccaa
     2041 tccggcaaac gcactggctg gtactctgcg cgctgattac gctgacagcc tgaccgaaaa
     2101 cggtacccac ggttctgatt ccgtcgaatc tgccgctcgc gaaatcgctt atttctttgg
     2161 cgaaggcgaa gtgtgcccgc gcacccgtta agtcgacctg cagccaagct taattagctg
     2221 agcttggact cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca
     2281 gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagctag cttggcgaga
     2341 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
     2401 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
     2461 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
     2521 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
     2581 tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
     2641 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     2701 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
     2761 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     2821 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
     2881 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     2941 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     3001 gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc ttaaacgcct
     3061 ggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt cgaaagactg
     3121 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     3181 ctctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3241 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3301 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     3361 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     3421 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     3481 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3541 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3601 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3661 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3721 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3781 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3841 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gta
//
