LOCUS       sequence-275252-        6878 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 6878)
  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 6878)
  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: pET21a-LysRS-His
FEATURES             Location/Qualifiers
     source          1..6878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(26..614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(106..125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(785..1576,1577..1645))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1408..1427
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1646..1750)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1776..2231)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1913..1934)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2125..2144
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2268..2315)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2311..2329
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2382..2399)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3928..3950
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3965..3989
                     /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)."
     primer_bind     complement(3989..4008)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3990..4008)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4092..4111)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4317..4394
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4395..5477
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4414..4433)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6286..6477
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6471..6493)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6579..6718
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6634..6653
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6733..6750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
       61 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      121 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      181 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      241 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      301 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      361 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      421 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
      481 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      541 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      601 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      661 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
      721 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
      781 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
      841 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
      901 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
      961 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1021 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1081 gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1141 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1201 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1261 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1321 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1381 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1441 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1501 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     1561 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     1621 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     1681 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1741 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     1801 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     1861 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     1921 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     1981 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     2041 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2101 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     2161 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     2221 acagggcgcg tcccattcgc caatccggat atagttcctc ctttcagcaa aaaacccctc
     2281 aagacccgtt tagaggcccc aaggggttat gctagttatt gctcagcggt ggcagcagcc
     2341 aactcagctt cctttcgggc tttgttagca gccggatctc agtggtggtg gtggtggtgc
     2401 tcgagtttct gtgggcgcat cgccgggaag agaataacgt cgcgaatagt atggctgtta
     2461 gtaaacagca taatcattcg gtcgatacca atacccagac cagcggttgg cggcagacca
     2521 tattccagcg cagtcacgta atcttcgtca tagaacatgg cttcgtcgtc acctgcagct
     2581 ttagcattaa cctgttcctg gaaacgttca gcctgatctt ctgcgtcgtt taattcgcta
     2641 aaaccattac cgatttcacg accaccgatg aagaattcaa aacggtcggt gatttccggg
     2701 ttaacatcat tacggcgtgc cagcggggac acttctgccg gatattccgt aataaaggtt
     2761 ggctgaatca gatgtgcttc tgccacttca tcaaagatct ctgtgacaat acgtcccaac
     2821 ccccagcttt tctctaccgt aataccgata gattcagcta atgctttagc agcatcaaaa
     2881 ttatccaggt cggccatatc ggtttctgga cgatattttt tgattgcttc gcgcatggtg
     2941 agtttttcaa acggtttgcc gaaatcaaac acatgctcgc cataagtgac tttagtggta
     3001 cccagaacct cttgtgccag ggtgcggaac agtgactctg tcagttcaat caaatcgtgg
     3061 taatccgcat acgccatgta gagttccatc attgtgaact caggattatg gcgaacagaa
     3121 ataccttcat tacggaagtt acggttgatt tcgaataccc gttcaaaacc gcctacaacc
     3181 agacgtttca gatacagctc cggcgcgata cgcagataca tatctaaatc cagagcatta
     3241 tgatgggtaa taaacgggcg agcagatgcc ccacctggaa ttacctgcat catcggggtt
     3301 tctacttcca taaagccgcg cgcgaccatg aattgacgga tagcggccag aatttttgaa
     3361 cggacaacaa acgtttgacg ggatttatcg ttagcgatga ggtccagata acgttgacga
     3421 taacggactt cctgatcctg cagaccatgg aatttatctg gtaaaggacg tagtgcttta
     3481 gtcagcaggc gcagctcagt acagtgaatg gaaagctcac ccgtttgcgt cttaaacagc
     3541 gtaccgcggg caccgataat gtcacccaga tcccattttt taaactgatc gttataaaca
     3601 ccttctggca ggctatctct tgcaacgtac agttgaatac ggccaccgac atcctgcaac
     3661 gttacaaagg aggctttccc catgatacga cgggtcatca ttcggccagc aaccgagact
     3721 tcaatgttta aggattccag ttcctggtta tccttcgcat caaactcttc gtgcaactgg
     3781 tcagaggtat ggtcgcggcg aaaatcattg ggaaacgcca caccttgctg acgtagtgcc
     3841 gccagttttt cgcggcgatt tctcagttca tcgttaaaat caatagcctc attggctccc
     3901 cgtgtttctt gttcagacat atgtatatct ccttcttaaa gttaaacaaa attatttcta
     3961 gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat
     4021 cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     4081 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     4141 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     4201 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     4261 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     4321 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     4381 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     4441 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     4501 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     4561 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     4621 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4681 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4741 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4801 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     4861 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     4921 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     4981 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     5041 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     5101 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     5161 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     5221 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     5281 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     5341 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     5401 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5461 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     5521 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     5581 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     5641 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     5701 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     5761 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     5821 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     5881 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     5941 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     6001 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     6061 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     6121 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     6181 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     6241 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     6301 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     6361 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     6421 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     6481 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     6541 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     6601 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     6661 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     6721 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6781 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6841 gaacatgtga gcaaaaggcc agcaaaaggc caggaacc
//
