LOCUS       sequence-258600-        6776 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 6776)
  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 6776)
  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-GluRS-His
FEATURES             Location/Qualifiers
     source          1..6776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(778..1860)
                     /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     1822..1841
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1861..1938)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2144..2163
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2247..2266
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2247..2265
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2266..2290
                     /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             2305..2327
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3754..3771
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3824..3842)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3838..3885
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3922..4377
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4009..4028)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4219..4240
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4403..4507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4508..4576,4577..5368)
                     /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     complement(4726..4745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5539..6127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6028..6047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6281..6298
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6313..6452
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6378..6397)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6538..6560
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6554..6745)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
       61 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      121 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      181 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      241 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      301 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      361 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      421 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      481 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      541 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      601 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      661 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      721 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
      781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      841 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      901 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      961 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1021 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1081 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1141 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1201 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1261 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1321 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1381 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1441 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1501 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1561 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1621 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1681 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1741 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1801 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1861 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1921 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1981 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     2041 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     2101 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     2161 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     2221 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     2281 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgaaa
     2341 atcaaaactc gcttcgcgcc aagcccaaca ggctatctgc acgttggcgg cgcgcgtact
     2401 gctctttact cctggctttt tgcacgtaac cacggcggtg agttcgtgct gcgtattgaa
     2461 gacaccgatc ttgagcgttc cacgccggaa gctatcgaag ccattatgga tggcatgaac
     2521 tggctgagcc tggagtggga tgaaggtccg tactaccaga ccaaacgttt tgatcgctac
     2581 aacgcggtga tcgatcagat gctggaagag ggcactgctt ataaatgcta ttgctctaaa
     2641 gagcgcctgg aagcgctgcg cgaagagcaa atggcgaaag gtgagaagcc gcgttatgac
     2701 ggtcgctgcc gccacagcca tgagcatcat gctgatgatg aaccgtgtgt tgtacgtttt
     2761 gctaacccgc aggaaggttc tgttgttttt gacgatcaga tccgtggtcc gatcgagttc
     2821 agcaaccagg aactggacga tcttattatc cgccgtaccg atggttcccc aacctataac
     2881 ttctgtgtgg ttgtcgatga ctgggatatg gaaatcaccc acgttatccg tggcgaagac
     2941 catatcaaca acacgccacg ccagatcaac attcttaagg ccctgaaagc gccggtgccg
     3001 gtttacgcgc acgtttctat gatcaatggc gatgacggta aaaaactgtc caaacgtcac
     3061 ggggcagtca gcgtaatgca gtatcgtgat gacggttatt tgccagaagc actgctgaac
     3121 tatctggtgc gtctgggctg gtcccacggc gatcaggaaa tcttcactcg tgaagagatg
     3181 atcaaatact tcactttgaa tgccgtcagc aaatctgcca gtgcgttcaa caccgacaag
     3241 ctgctgtggc tgaaccatca ctacattaac gcgctgccgc cggagtatgt tgctactcac
     3301 ttacagtggc acattgagca ggaaaatatc gatacccgta acggcccgca gctggctgat
     3361 ctggtgaaac tgctgggcga acgctgcaag acgctgaaag agatggcaca gagctgccgt
     3421 tatttctacg aagattttgc tgagttcgat gccgacgccg cgaaaaaaca tctgcgtccg
     3481 gtagcgcgtc agccgctgga agtggttcgt gacaaactgg ccgcgattac tgactggacc
     3541 gctgaaaacg ttcatcacgc tattcaggcg acggcggatg agctggaagt gggtatgggt
     3601 aaagttggta tgccgctgcg tgtcgccgta accggtgcgg ggcagtctcc agcactggat
     3661 gttaccgttc acgcaattgg taagacccgc agtatcgagc gtatcaacaa agcgctggat
     3721 tttattgctg aacgcgaaaa tcagcagctc gagcaccacc accaccacca ctgagatccg
     3781 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     3841 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     3901 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3961 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4021 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4081 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4141 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4201 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4261 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4321 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     4381 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4441 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4501 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4561 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4621 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4681 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4741 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4801 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4861 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4921 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4981 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5041 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5101 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5161 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5221 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5281 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5341 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5401 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5461 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5521 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5581 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5641 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     5701 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5761 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5821 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5881 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5941 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6001 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6061 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6121 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6181 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6241 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6301 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6361 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     6421 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     6481 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     6541 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     6601 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     6661 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     6721 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttc
//
